Microorganisms and methods for the biosynthesis of p-toluate and terephthalate

ABSTRACT

The invention provides non-naturally occurring microbial organisms having a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway, p-toluate pathway, and/or terephthalate pathway. The invention additionally provides methods of using such organisms to produce (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway, p-toluate pathway or terephthalate pathway.

BACKGROUND OF THE INVENTION

The present invention relates generally to biosynthetic processes, andmore specifically to organisms having p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate biosynthetic capability.

Terephthalate (also known as terephthalic acid and PTA) is the immediateprecursor of polyethylene terepthalate (PET), used to make clothing,resins, plastic bottles and even as a poultry feed additive. Nearly allPTA is produced from para-xylene by oxidation in air in a process knownas the Mid Century Process. This oxidation is conducted at hightemperature in an acetic acid solvent with a catalyst composed of cobaltand/or manganese salts. Para-xylene is derived from petrochemicalsources and is formed by high severity catalytic reforming of naphtha.Xylene is also obtained from the pyrolysis gasoline stream in a naphthasteam cracker and by toluene disproportion.

Cost-effective methods for generating renewable PTA have not yet beendeveloped to date. PTA, toluene and other aromatic precursors arenaturally degraded by some bacteria. However, these degradation pathwaystypically involve monooxygenases that operate irreversibly in thedegradative direction. Hence, biosynthetic pathways for PTA are severelylimited by the properties of known enzymes to date.

A promising precursor for PTA is p-toluate, also known asp-methylbenzoate. P-Toluate is an intermediate in some industrialprocesses for the oxidation of p-xylene to PTA. It is also anintermediate for polymer stabilizers, pesticides, light sensitivecompounds, animal feed supplements and other organic chemicals. Onlyslightly soluble in aqueous solution, p-toluate is a solid atphysiological temperatures, with a melting point of 275° C. Microbialcatalysts for synthesizing this compound from sugar feedstocks have notbeen described to date.

Thus, there exists a need for alternative methods for effectivelyproducing commercial quantities of compounds such as p-toluate orterephthalate. The present invention satisfies this need and providesrelated advantages as well.

SUMMARY OF THE INVENTION

The invention provides non-naturally occurring microbial organismshaving a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway, p-toluatepathway, and/or terephthalate pathway. The invention additionallyprovides methods of using such organisms to produce(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway, p-toluate pathwayor terephthalate pathway.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic depiction of an exemplary pathway to(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate (2H3M4OP) fromglyceraldehyde-3-phosphate and pyruvate. G3P isglyceraldehyde-3-phosphate, DXP is 1-deoxy-D-xylulose-5-phosphate and2ME4P is C-methyl-D-erythritol-4-phosphate. Enzymes are (A) DXPsynthase; (B) DXP reductoisomerase; and (C) 2ME4P dehydratase.

FIG. 2 shows a schematic depiction of an exemplary alternate shikimatepathway to p-toluate. Enzymes are: (A) 2-dehydro-3-deoxyphosphoheptonatesynthase; (B) 3-dehydroquinate synthase; (C) 3-dehydroquinatedehydratase; (D) shikimate dehydrogenase; (E) Shikimate kinase; (F)3-phosphoshikimate-2-carboxyvinyltransferase; (G) chorismate synthase;and (H) chorismate lyase. Compounds are: (1)(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate; (2)2,4-dihydroxy-5-methyl-6-[(phosphonooxy)methyl]oxane-2-carboxylate; (3)1,3-dihydroxy-4-methyl-5-oxocyclohexane-1-carboxylate; (4)5-hydroxy-4-methyl-3-oxocyclohex-1-ene-1-carboxylate; (5)3,5-dihydroxy-4-methylcyclohex-1-ene-1-carboxylate; (6)5-hydroxy-4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate; (7)5-[(1-carboxyeth-1-en-1-yl)oxy]-4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate;(8)3-[(1-carboxyeth-1-en-1-yl)oxy]-4-methylcyclohexa-1,5-diene-1-carboxylate;and (9) p-toluate.

FIG. 3 shows an exemplary pathway for conversion of p-toluate toterephthalic acid (PTA). Reactions A, B and C are catalyzed by p-toluatemethyl-monooxygenase reductase, 4-carboxybenzyl alcohol dehydrogenaseand 4-carboxybenzyl aldehyde dehydrogenase, respectively. The compoundsshown are (1) p-toluic acid; (2) 4-carboxybenzyl alcohol; (3)4-carboxybenzaldehyde and (4) terephthalic acid.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to the design and production of cellsand organisms having biosynthetic production capabilities for p-toluate,terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate. Theresults described herein indicate that metabolic pathways can bedesigned and recombinantly engineered to achieve the biosynthesis ofp-toluate, terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonatein Escherichia coli and other cells or organisms. Biosyntheticproduction of p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate can be confirmed byconstruction of strains having the designed metabolic genotype. Thesemetabolically engineered cells or organisms also can be subjected toadaptive evolution to further augment p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate biosynthesis, includingunder conditions approaching theoretical maximum growth.

The shikimate biosynthesis pathway in E. coli convertserythrose-4-phosphate to chorismate, an important intermediate thatleads to the biosynthesis of many essential metabolites including4-hydroxybenzoate. 4-Hydroxybenzoate is structurally similar top-toluate, an industrial precursor of terephthalic acid. As disclosedherein, shikimate pathway enzymes are utilized to accept the alternatesubstrate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate (2H3M4OP) andtransform it to p-toluate. In addition, a pathway is used to synthesizethe 2H3M4OP precursor using enzymes from the non-mevalonate pathway forisoprenoid biosynthesis.

Disclosed herein are strategies for engineering a microorganism toproduce renewable p-toluate or terephthalate (PTA) from carbohydratefeedstocks. First, glyceraldehyde-3-phosphate (G3P) and pyruvate areconverted to 2-hydroxy-3-methyl-4-oxobutoxy)phosphonate (2H3M4OP) inthree enzymatic steps (see Example I and FIG. 1). The 2H3M4OPintermediate is subsequently transformed to p-toluate by enzymes in theshikimate pathway (see Example II and FIG. 2). P-Toluate can be furtherconverted to PTA by a microorganism (see Example III and FIG. 3).

The conversion of G3P to p-toluate requires one ATP, two reducingequivalents (NAD(P)H), and two molecules of phosphoenolpyruvate,according to net reaction below.

G3P+2 PEP+ATP+2 NAD(P)H+2 H⁺→p-Toluate+4 Pi+ADP+2 NAD(P)⁺+CO₂+H₂O

An additional ATP is required to synthesize G3P from glucose. Themaximum theoretical p-toluate yield is 0.67 mol/mol (0.51 g/g) fromglucose minus carbon required for energy. Under the assumption that 2ATPs are consumed per p-toluate molecule synthesized, the predictedp-toluate yield from glucose is 0.62 mol/mol (0.46 g/g) p-toluate.

If p-toluate is further converted to PTA by enzymes as described inExample III, the predicted PTA yield from glucose is 0.64 mol/mol (0.58g/g). In this case, the oxidation of p-toluate to PTA generates anadditional net reducing equivalent according to the net reaction:

p-toluate+O₂+NAD⁺→PTA+NADH+2 H⁺

Enzyme candidates for catalyzing each step of the proposed pathways aredescribed in the following sections.

As used herein, the term “non-naturally occurring” when used inreference to a microbial organism or microorganism of the invention isintended to mean that the microbial organism has at least one geneticalteration not normally found in a naturally occurring strain of thereferenced species, including wild-type strains of the referencedspecies. Genetic alterations include, for example, modificationsintroducing expressible nucleic acids encoding metabolic polypeptides,other nucleic acid additions, nucleic acid deletions and/or otherfunctional disruption of the microbial organism's genetic material. Suchmodifications include, for example, coding regions and functionalfragments thereof, for heterologous, homologous or both heterologous andhomologous polypeptides for the referenced species. Additionalmodifications include, for example, non-coding regulatory regions inwhich the modifications alter expression of a gene or operon. Exemplarymetabolic polypeptides include enzymes or proteins within a p-toluate,terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonatebiosynthetic pathway.

A metabolic modification refers to a biochemical reaction that isaltered from its naturally occurring state. Therefore, non-naturallyoccurring microorganisms can have genetic modifications to nucleic acidsencoding metabolic polypeptides, or functional fragments thereof.Exemplary metabolic modifications are disclosed herein.

As used herein, the term “isolated” when used in reference to amicrobial organism is intended to mean an organism that is substantiallyfree of at least one component as the referenced microbial organism isfound in nature. The term includes a microbial organism that is removedfrom some or all components as it is found in its natural environment.The term also includes a microbial organism that is removed from some orall components as the microbial organism is found in non-naturallyoccurring environments. Therefore, an isolated microbial organism ispartly or completely separated from other substances as it is found innature or as it is grown, stored or subsisted in non-naturally occurringenvironments. Specific examples of isolated microbial organisms includepartially pure microbes, substantially pure microbes and microbescultured in a medium that is non-naturally occurring.

As used herein, the terms “microbial,” “microbial organism” or“microorganism” are intended to mean any organism that exists as amicroscopic cell that is included within the domains of archaea,bacteria or eukarya. Therefore, the term is intended to encompassprokaryotic or eukaryotic cells or organisms having a microscopic sizeand includes bacteria, archaea and eubacteria of all species as well aseukaryotic microorganisms such as yeast and fungi. The term alsoincludes cell cultures of any species that can be cultured for theproduction of a biochemical.

As used herein, the term “CoA” or “coenzyme A” is intended to mean anorganic cofactor or prosthetic group (nonprotein portion of an enzyme)whose presence is required for the activity of many enzymes (theapoenzyme) to form an active enzyme system. Coenzyme A functions incertain condensing enzymes, acts in acetyl or other acyl group transferand in fatty acid synthesis and oxidation, pyruvate oxidation and inother acetylation.

As used herein, the term “(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,”abbreviated herein as 2H3M4OP, has the chemical formula as shown inFIG. 1. Such a compound can also be described as 3-hydroxy-2-methylbutanal-4-phosphate.

As used herein, the term “p-toluate,” having the molecular formulaC₈H₇O₂ ⁻ (see FIG. 2, compound 9)(IUPAC name 4-methylbenzoate) is theionized form of p-toluic acid, and it is understood that p-toluate andp-toluic acid can be used interchangeably throughout to refer to thecompound in any of its neutral or ionized forms, including any saltforms thereof. It is understood by those skilled understand that thespecific form will depend on the pH.

As used herein, the term “terephthalate,” having the molecular formulaC₈H₄O₄ ⁻² (see FIG. 3, compound 4)(IUPAC name terephthalate) is theionized form of terephthalic acid, also referred to as p-phthalic acidor PTA, and it is understood that terephthalate and terephthalic acidcan be used interchangeably throughout to refer to the compound in anyof its neutral or ionized forms, including any salt forms thereof It isunderstood by those skilled understand that the specific form willdepend on the pH.

As used herein, the term “substantially anaerobic” when used inreference to a culture or growth condition is intended to mean that theamount of oxygen is less than about 10% of saturation for dissolvedoxygen in liquid media. The term also is intended to include sealedchambers of liquid or solid medium maintained with an atmosphere of lessthan about 1% oxygen.

“Exogenous” as it is used herein is intended to mean that the referencedmolecule or the referenced activity is introduced into the hostmicrobial organism. The molecule can be introduced, for example, byintroduction of an encoding nucleic acid into the host genetic materialsuch as by integration into a host chromosome or as non-chromosomalgenetic material such as a plasmid. Therefore, the term as it is used inreference to expression of an encoding nucleic acid refers tointroduction of the encoding nucleic acid in an expressible form intothe microbial organism. When used in reference to a biosyntheticactivity, the term refers to an activity that is introduced into thehost reference organism. The source can be, for example, a homologous orheterologous encoding nucleic acid that expresses the referencedactivity following introduction into the host microbial organism.Therefore, the term “endogenous” refers to a referenced molecule oractivity that is present in the host. Similarly, the term when used inreference to expression of an encoding nucleic acid refers to expressionof an encoding nucleic acid contained within the microbial organism. Theterm “heterologous” refers to a molecule or activity derived from asource other than the referenced species whereas “homologous” refers toa molecule or activity derived from the host microbial organism.Accordingly, exogenous expression of an encoding nucleic acid of theinvention can utilize either or both a heterologous or homologousencoding nucleic acid.

It is understood that when more than one exogenous nucleic acid isincluded in a microbial organism that the more than one exogenousnucleic acids refers to the referenced encoding nucleic acid orbiosynthetic activity, as discussed above. It is further understood, asdisclosed herein, that such more than one exogenous nucleic acids can beintroduced into the host microbial organism on separate nucleic acidmolecules, on polycistronic nucleic acid molecules, or a combinationthereof, and still be considered as more than one exogenous nucleicacid. For example, as disclosed herein a microbial organism can beengineered to express two or more exogenous nucleic acids encoding adesired pathway enzyme or protein. In the case where two exogenousnucleic acids encoding a desired activity are introduced into a hostmicrobial organism, it is understood that the two exogenous nucleicacids can be introduced as a single nucleic acid, for example, on asingle plasmid, on separate plasmids, can be integrated into the hostchromosome at a single site or multiple sites, and still be consideredas two exogenous nucleic acids. Similarly, it is understood that morethan two exogenous nucleic acids can be introduced into a host organismin any desired combination, for example, on a single plasmid, onseparate plasmids, can be integrated into the host chromosome at asingle site or multiple sites, and still be considered as two or moreexogenous nucleic acids, for example three exogenous nucleic acids.Thus, the number of referenced exogenous nucleic acids or biosyntheticactivities refers to the number of encoding nucleic acids or the numberof biosynthetic activities, not the number of separate nucleic acidsintroduced into the host organism.

The non-naturally occurring microbial organisms of the invention cancontain stable genetic alterations, which refers to microorganisms thatcan be cultured for greater than five generations without loss of thealteration. Generally, stable genetic alterations include modificationsthat persist greater than 10 generations, particularly stablemodifications will persist more than about 25 generations, and moreparticularly, stable genetic modifications will be greater than 50generations, including indefinitely.

Those skilled in the art will understand that the genetic alterations,including metabolic modifications exemplified herein, are described withreference to a suitable host organism such as E. coli and theircorresponding metabolic reactions or a suitable source organism fordesired genetic material such as genes for a desired metabolic pathway.However, given the complete genome sequencing of a wide variety oforganisms and the high level of skill in the area of genomics, thoseskilled in the art will readily be able to apply the teachings andguidance provided herein to essentially all other organisms. Forexample, the E. coli metabolic alterations exemplified herein canreadily be applied to other species by incorporating the same oranalogous encoding nucleic acid from species other than the referencedspecies. Such genetic alterations include, for example, geneticalterations of species homologs, in general, and in particular,orthologs, paralogs or nonorthologous gene displacements.

An ortholog is a gene or genes that are related by vertical descent andare responsible for substantially the same or identical functions indifferent organisms. For example, mouse epoxide hydrolase and humanepoxide hydrolase can be considered orthologs for the biologicalfunction of hydrolysis of epoxides. Genes are related by verticaldescent when, for example, they share sequence similarity of sufficientamount to indicate they are homologous, or related by evolution from acommon ancestor. Genes can also be considered orthologs if they sharethree-dimensional structure but not necessarily sequence similarity, ofa sufficient amount to indicate that they have evolved from a commonancestor to the extent that the primary sequence similarity is notidentifiable. Genes that are orthologous can encode proteins withsequence similarity of about 25% to 100% amino acid sequence identity.Genes encoding proteins sharing an amino acid similarity less that 25%can also be considered to have arisen by vertical descent if theirthree-dimensional structure also shows similarities. Members of theserine protease family of enzymes, including tissue plasminogenactivator and elastase, are considered to have arisen by verticaldescent from a common ancestor.

Orthologs include genes or their encoded gene products that through, forexample, evolution, have diverged in structure or overall activity. Forexample, where one species encodes a gene product exhibiting twofunctions and where such functions have been separated into distinctgenes in a second species, the three genes and their correspondingproducts are considered to be orthologs. For the production of abiochemical product, those skilled in the art will understand that theorthologous gene harboring the metabolic activity to be introduced ordisrupted is to be chosen for construction of the non-naturallyoccurring microorganism. An example of orthologs exhibiting separableactivities is where distinct activities have been separated intodistinct gene products between two or more species or within a singlespecies. A specific example is the separation of elastase proteolysisand plasminogen proteolysis, two types of serine protease activity, intodistinct molecules as plasminogen activator and elastase. A secondexample is the separation of mycoplasma 5′-3′ exonuclease and DrosophilaDNA polymerase III activity. The DNA polymerase from the first speciescan be considered an ortholog to either or both of the exonuclease orthe polymerase from the second species and vice versa.

In contrast, paralogs are homologs related by, for example, duplicationfollowed by evolutionary divergence and have similar or common, but notidentical functions. Paralogs can originate or derive from, for example,the same species or from a different species. For example, microsomalepoxide hydrolase (epoxide hydrolase I) and soluble epoxide hydrolase(epoxide hydrolase II) can be considered paralogs because they representtwo distinct enzymes, co-evolved from a common ancestor, that catalyzedistinct reactions and have distinct functions in the same species.Paralogs are proteins from the same species with significant sequencesimilarity to each other suggesting that they are homologous, or relatedthrough co-evolution from a common ancestor. Groups of paralogousprotein families include HipA homologs, luciferase genes, peptidases,and others.

A nonorthologous gene displacement is a nonorthologous gene from onespecies that can substitute for a referenced gene function in adifferent species. Substitution includes, for example, being able toperform substantially the same or a similar function in the species oforigin compared to the referenced function in the different species.Although generally, a nonorthologous gene displacement will beidentifiable as structurally related to a known gene encoding thereferenced function, less structurally related but functionally similargenes and their corresponding gene products nevertheless will still fallwithin the meaning of the term as it is used herein. Functionalsimilarity requires, for example, at least some structural similarity inthe active site or binding region of a nonorthologous gene productcompared to a gene encoding the function sought to be substituted.Therefore, a nonorthologous gene includes, for example, a paralog or anunrelated gene.

Therefore, in identifying and constructing the non-naturally occurringmicrobial organisms of the invention having p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate biosynthetic capability,those skilled in the art will understand with applying the teaching andguidance provided herein to a particular species that the identificationof metabolic modifications can include identification and inclusion orinactivation of orthologs. To the extent that paralogs and/ornonorthologous gene displacements are present in the referencedmicroorganism that encode an enzyme catalyzing a similar orsubstantially similar metabolic reaction, those skilled in the art alsocan utilize these evolutionally related genes.

Orthologs, paralogs and nonorthologous gene displacements can bedetermined by methods well known to those skilled in the art. Forexample, inspection of nucleic acid or amino acid sequences for twopolypeptides will reveal sequence identity and similarities between thecompared sequences. Based on such similarities, one skilled in the artcan determine if the similarity is sufficiently high to indicate theproteins are related through evolution from a common ancestor.Algorithms well known to those skilled in the art, such as Align, BLAST,Clustal W and others compare and determine a raw sequence similarity oridentity, and also determine the presence or significance of gaps in thesequence which can be assigned a weight or score. Such algorithms alsoare known in the art and are similarly applicable for determiningnucleotide sequence similarity or identity. Parameters for sufficientsimilarity to determine relatedness are computed based on well knownmethods for calculating statistical similarity, or the chance of findinga similar match in a random polypeptide, and the significance of thematch determined. A computer comparison of two or more sequences can, ifdesired, also be optimized visually by those skilled in the art. Relatedgene products or proteins can be expected to have a high similarity, forexample, 25% to 100% sequence identity. Proteins that are unrelated canhave an identity which is essentially the same as would be expected tooccur by chance, if a database of sufficient size is scanned (about 5%).Sequences between 5% and 24% may or may not represent sufficienthomology to conclude that the compared sequences are related. Additionalstatistical analysis to determine the significance of such matches giventhe size of the data set can be carried out to determine the relevanceof these sequences.

Exemplary parameters for determining relatedness of two or moresequences using the BLAST algorithm, for example, can be as set forthbelow. Briefly, amino acid sequence alignments can be performed usingBLASTP version 2.0.8 (Jan. 5, 1999) and the following parameters:Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1; x_dropoff: 50;expect: 10.0; wordsize: 3; filter: on. Nucleic acid sequence alignmentscan be performed using BLASTN version 2.0.6 (Sep. 16, 1998) and thefollowing parameters: Match: 1; mismatch: −2; gap open: 5; gapextension: 2; x_dropoff: 50; expect: 10.0; wordsize: 11; filter: off.Those skilled in the art will know what modifications can be made to theabove parameters to either increase or decrease the stringency of thecomparison, for example, and determine the relatedness of two or moresequences.

The invention provides a non-naturally occurring microbial organism,comprising a microbial organism having a(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway comprising at leastone exogenous nucleic acid encoding a(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway enzyme expressed ina sufficient amount to produce(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, the(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway comprising2-C-methyl-D-erythritol-4-phosphate dehydratase (see Example I and FIG.1, step C). A non-naturally occurring microbial organism comprising a(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway can further comprise1-deoxyxylulose-5-phosphate synthase or 1-deoxy-D-xylulose-5-phosphatereductoisomerase (see Example I and FIG. 1, steps A and B). Thus, a(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate can comprise 52-C-methyl-D-erythritol-4-phosphate dehydratase,1-deoxyxylulose-5-phosphate synthase and 1-deoxy-D-xylulose-5-phosphatereductoisomerase.

The invention also provides a non-naturally occurring microbialorganism, comprising a microbial organism having a p-toluate pathwaycomprising at least one exogenous nucleic acid encoding a p-toluatepathway enzyme expressed in a sufficient amount to produce p-toluate,the p-toluate pathway comprising 2-dehydro-3-deoxyphosphoheptonatesynthase; 3-dehydroquinate synthase; 3-dehydroquinate dehydratase;shikimate dehydrogenase; shikimate kinase;3-phosphoshikimate-2-carboxyvinyltransferase; chorismate synthase; orchorismate lyase (see Example II and FIG. 2, steps A-H). A non-naturallyoccurring microbial organism having a p-toluate pathway can furthercomprise a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway (FIG. 1).A (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway can comprise, forexample, 2-C-methyl-D-erythritol-4-phosphate dehydratase,1-deoxyxylulose-5-phosphate synthase or 1-deoxy-D-xylulose-5-phosphatereductoisomerase (FIG. 1).

The invention additionally provides a non-naturally occurring microbialorganism, comprising a microbial organism having a terephthalate pathwaycomprising at least one exogenous nucleic acid encoding a terephthalatepathway enzyme expressed in a sufficient amount to produceterephthalate, the terephthalate pathway comprising p-toluatemethyl-monooxygenase reductase; 4-carboxybenzyl alcohol dehydrogenase;or 4-carboxybenzyl aldehyde dehydrogenase (see Example III and FIG. 3).Such an organism containing a terephthalate pathway can additionallycomprise a p-toluate pathway, wherein the p-toluate pathway comprises2-dehydro-3-deoxyphosphoheptonate synthase; 3-dehydroquinate synthase;3-dehydroquinate dehydratase; shikimate dehydrogenase; shikimate kinase;3-phosphoshikimate-2-carboxyvinyltransferase; chorismate synthase; orchorismate lyase (see Examples II and III and FIGS. 2 and 3). Such anon-naturally occurring microbialorganism having a terephthalate pathwayand a p-toluate pathway can further comprise a(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway (see Example I andFIG. 1). A (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway cancomprise, for example, 2-C-methyl-D-erythritol-4-phosphate dehydratase,1-deoxyxylulose-5-phosphate synthase or 1-deoxy-D-xylulose-5-phosphatereductoisomerase (see Example I and FIG. 1).

In an additional embodiment, the invention provides a non-naturallyoccurring microbial organism having a p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway, wherein thenon-naturally occurring microbial organism comprises at least oneexogenous nucleic acid encoding an enzyme or protein that converts asubstrate to a product. For example, in a(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway, the substrates andproducts can be selected from the group consisting ofglyceraldehyde-3-phosphate and pyruvate to1-deoxy-D-xylulose-5-phosphate; 1-deoxy-D-xylulose-5-phosphate toC-methyl-D-erythritol-4-phosphate; and C-methyl-D-erythritol-4-phosphateto (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate (see Example I and FIG.1). In another embodiment, a p-toluate pathway can comprise substratesand products selected from (2-hydroxy-3-methyl-4-oxobutoxy)phosphonateto 2,4-dihydroxy-5-methyl-6-[(phosphonooxy)methyl]oxane-2-carboxylate;2,4-dihydroxy-5-methyl-6-[(phosphonooxy)methyl]oxane-2-carboxylate to1,3-dihydroxy-4-methyl-5-oxocyclohexane-1-carboxylate;1,3-dihydroxy-4-methyl-5-oxocyclohexane-1-carboxylate to5-hydroxy-4-methyl-3-oxocyclohex-1-ene-1-carboxylic acid;5-hydroxy-4-methyl-3-oxocyclohex-1-ene-1-carboxylic acid to3,5-dihydroxy-4-methylcyclohex-1-ene-1-carboxylate;3,5-dihydroxy-4-methylcyclohex-1-ene-1-carboxylate to5-hydroxy-4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate;5-hydroxy-4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate to5-[(1-carboxyeth-1-en-1-yl)oxy]-4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate;5-[(1-carboxyeth-1-en-1-yl)oxy]-4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-carboxylateto3-[(1-carboxyeth-1-en-1-yl)oxy]-4-methylcyclohexa-1,5-diene-1-carboxylate;and3-[(1-carboxyeth-1-en-1-yl)oxy]-4-methylcyclohexa-1,5-diene-1-carboxylateto p-toluate (see Example II and FIG. 2). In still another embodiment, aterephthalate pathway can comprise substrates and products selected fromp-toluate to 4-carboxybenzyl alcohol; 4-carboxybenzyl alcohol to4-carboxybenzaldehyde; and 4-carboxybenzaldehyde to and terephthalicacid (see Example III and FIG. 3). One skilled in the art willunderstand that these are merely exemplary and that any of thesubstrate-product pairs disclosed herein suitable to produce a desiredproduct and for which an appropriate activity is available for theconversion of the substrate to the product can be readily determined byone skilled in the art based on the teachings herein. Thus, theinvention provides a non-naturally occurring microbial organismcontaining at least one exogenous nucleic acid encoding an enzyme orprotein, where the enzyme or protein converts the substrates andproducts of a p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway, such as that shownin FIGS. 1-3.

While generally described herein as a microbial organism that contains ap-toluate, terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonatepathway, it is understood that the invention additionally provides anon-naturally occurring microbial organism comprising at least oneexogenous nucleic acid encoding a p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway enzyme expressed ina sufficient amount to produce an intermediate of a p-toluate,terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway.For example, as disclosed herein, a(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway is exemplified inFIG. 1 (see Example I). Therefore, in addition to a microbial organismcontaining a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway thatproduces (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, the inventionadditionally provides a non-naturally occurring microbial organismcomprising at least one exogenous nucleic acid encoding a(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway enzyme, where themicrobial organism produces a(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway intermediate, forexample, 1-deoxy-D-xylulose-5-phosphate orC-methyl-D-erythritol-4-phosphate. Similarly, the invention alsoprovides a non-naturally occurring microbial organism containing ap-toluate pathway that produces p-toluate, wherein the non-naturallyoccurring microbial organism comprises at least one exogenous nucleicacid encoding a p-toluate pathway enzyme, where the microbial organismproduces a p-toluate pathway intermediate, for example,2,4-dihydroxy-5-methyl-6-[(phosphonooxy)methyl]oxane-2-carboxyl ate,1,3-dihydroxy-4-methyl-5-oxocyclohexane-1-carboxylate,5-hydroxy-4-methyl-3-oxocyclohex-1-ene-1-carboxylate,3,5-dihydroxy-4-methylcyclohex-1-ene-1-carboxylate,5-hydroxy-4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate,5-[(1-carboxyeth-1-en-1-yl)oxy]-4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate,or3-[(1-carboxyeth-1-en-1-yl)oxy]-4-methylcyclohexa-1,5-diene-1-carboxylate.Further, the invention additionally provides a non-naturally occurringmicrobial organism containing a terephthalate pathway enzyme, where themicrobial organism produces a terephthalate pathway intermediate, forexample, 4-carboxybenzyl alcohol or 4-carboxybenzaldehyde.

It is understood that any of the pathways disclosed herein, as describedin the Examples and exemplified in the Figures, including the pathwaysof FIGS. 1-3, can be utilized to generate a non-naturally occurringmicrobial organism that produces any pathway intermediate or product, asdesired. As disclosed herein, such a microbial organism that produces anintermediate can be used in combination with another microbial organismexpressing downstream pathway enzymes to produce a desired product.However, it is understood that a non-naturally occurring microbialorganism that produces a p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway intermediate can beutilized to produce the intermediate as a desired product.

The invention is described herein with general reference to themetabolic reaction, reactant or product thereof, or with specificreference to one or more nucleic acids or genes encoding an enzymeassociated with or catalyzing, or a protein associated with, thereferenced metabolic reaction, reactant or product. Unless otherwiseexpressly stated herein, those skilled in the art will understand thatreference to a reaction also constitutes reference to the reactants andproducts of the reaction. Similarly, unless otherwise expressly statedherein, reference to a reactant or product also references the reaction,and reference to any of these metabolic constituents also references thegene or genes encoding the enzymes that catalyze or proteins involved inthe referenced reaction, reactant or product. Likewise, given the wellknown fields of metabolic biochemistry, enzymology and genomics,reference herein to a gene or encoding nucleic acid also constitutes areference to the corresponding encoded enzyme and the reaction itcatalyzes or a protein associated with the reaction as well as thereactants and products of the reaction.

The non-naturally occurring microbial organisms of the invention can beproduced by introducing expressible nucleic acids encoding one or moreof the enzymes or proteins participating in one or more p-toluate,terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonatebiosynthetic pathways. Depending on the host microbial organism chosenfor biosynthesis, nucleic acids for some or all of a particularp-toluate, terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonatebiosynthetic pathway can be expressed. For example, if a chosen host isdeficient in one or more enzymes or proteins for a desired biosyntheticpathway, then expressible nucleic acids for the deficient enzyme(s) orprotein(s) are introduced into the host for subsequent exogenousexpression. Alternatively, if the chosen host exhibits endogenousexpression of some pathway genes, but is deficient in others, then anencoding nucleic acid is needed for the deficient enzyme(s) orprotein(s) to achieve p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate biosynthesis. Thus, anon-naturally occurring microbial organism of the invention can beproduced by introducing exogenous enzyme or protein activities to obtaina desired biosynthetic pathway or a desired biosynthetic pathway can beobtained by introducing one or more exogenous enzyme or proteinactivities that, together with one or more endogenous enzymes orproteins, produces a desired product such as p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate.

Host microbial organisms can be selected from, and the non-naturallyoccurring microbial organisms generated in, for example, bacteria,yeast, fungus or any of a variety of other microorganisms applicable tofermentation processes. Exemplary bacteria include species selected fromEscherichia coli, Klebsiella oxytoca, Anaerobiospirillumsucciniciproducens, Actinobacillus succinogenes, Mannheimiasucciniciproducens, Rhizobium etli, Bacillus subtilis, Corynebacteriumglutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcuslactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridiumacetobutylicum, Pseudomonas fluorescens, and Pseudomonas putida.Exemplary yeasts or fungi include species selected from Saccharomycescerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis,Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Pichiapastoris, Rhizopus arrhizus, Rhizobus oryzae, and the like. E. coli is aparticularly useful host organisms since it is a well characterizedmicrobial organism suitable for genetic engineering. Other particularlyuseful host organisms include yeast such as Saccharomyces cerevisiae. Itis understood that any suitable microbial host organism can be used tointroduce metabolic and/or genetic modifications to produce a desiredproduct.

Depending on the p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate biosynthetic pathwayconstituents of a selected host microbial organism, the non-naturallyoccurring microbial organisms of the invention will include at least oneexogenously expressed p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway-encoding nucleicacid and up to all encoding nucleic acids for one or more p-toluate,terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonatebiosynthetic pathways. For example, p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate biosynthesis can beestablished in a host deficient in a pathway enzyme or protein throughexogenous expression of the corresponding encoding nucleic acid. In ahost deficient in all enzymes or proteins of a p-toluate, terephthalateor (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway, exogenousexpression of all enzyme or proteins in the pathway can be included,although it is understood that all enzymes or proteins of a pathway canbe expressed even if the host contains at least one of the pathwayenzymes or proteins. For example, exogenous expression of all enzymes orproteins in a pathway for production of p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate can be included. Forexample, all enzymes in a p-toluate pathway can be included, such as2-dehydro-3-deoxyphosphoheptonate synthase; 3-dehydroquinate synthase;3-dehydroquinate dehydratase; shikimate dehydrogenase; shikimate kinase;3-phosphoshikimate-2-carboxyvinyltransferase; chorismate synthase; andchorismate lyase. In addition, all enzymes in a terephthalate pathwaycan be included, such as p-toluate methyl-monooxygenase reductase;4-carboxybenzyl alcohol dehydrogenase; and 4-carboxybenzyl aldehydedehydrogenase. Furthermore, all enzymes in a(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway can be included,such as 2-C-methyl-D-erythritol-4-phosphate dehydratase,1-deoxyxylulose-5-phosphate synthase and 1-deoxy-D-xylulose-5-phosphatereductoisomerase.

Given the teachings and guidance provided herein, those skilled in theart will understand that the number of encoding nucleic acids tointroduce in an expressible form will, at least, parallel the p-toluate,terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathwaydeficiencies of the selected host microbial organism. Therefore, anon-naturally occurring microbial organism of the invention can haveone, two, three, four, five, six, seven, or eight, depending on theparticular pathway, that is, up to all nucleic acids encoding theenzymes or proteins constituting a p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate biosynthetic pathwaydisclosed herein. In some embodiments, the non-naturally occurringmicrobial organisms also can include other genetic modifications thatfacilitate or optimize p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate biosynthesis or that conferother useful functions onto the host microbial organism. One such otherfunctionality can include, for example, augmentation of the synthesis ofone or more of the p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway precursors such asglyceraldehyde-3-phosphate, pyruvate,(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate or p-toluate. Furthermore,as disclosed herein, multiple pathways can be included in a singleorganism such as the pathway to produce p-toluate (FIG. 2),terephthalate (FIGS. 3) and (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate(FIG. 1), as desired.

Generally, a host microbial organism is selected such that it producesthe precursor of a p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway, either as anaturally produced molecule or as an engineered product that eitherprovides de novo production of a desired precursor or increasedproduction of a precursor naturally produced by the host microbialorganism. For example, glyceraldehyde-3-phosphate andphosphoenolpyruvate are produced naturally in a host organism such as E.coli. A host organism can be engineered to increase production of aprecursor, as disclosed herein. In addition, a microbial organism thathas been engineered to produce a desired precursor can be used as a hostorganism and further engineered to express enzymes or proteins of ap-toluate, terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonatepathway.

In some embodiments, a non-naturally occurring microbial organism of theinvention is generated from a host that contains the enzymaticcapability to synthesize p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate. In this specific embodimentit can be useful to increase the synthesis or accumulation of ap-toluate, terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonatepathway product to, for example, drive p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway reactions towardp-toluate, terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonateproduction. Increased synthesis or accumulation can be accomplished by,for example, overexpression of nucleic acids encoding one or more of theabove-described p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway enzymes or proteins.Over expression the enzyme or enzymes and/or protein or proteins of thep-toluate, terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonatepathway can occur, for example, through exogenous expression of theendogenous gene or genes, or through exogenous expression of theheterologous gene or genes. Therefore, naturally occurring organisms canbe readily generated to be non-naturally occurring microbial organismsof the invention, for example, producing p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, through overexpression ofone, two, three, four, five, and so forth, that is, up to all nucleicacids encoding p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate biosynthetic pathway enzymesor proteins. In addition, a non-naturally occurring organism can begenerated by mutagenesis of an endogenous gene that results in anincrease in activity of an enzyme in the p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate biosynthetic pathway.

In particularly useful embodiments, exogenous expression of the encodingnucleic acids is employed. Exogenous expression confers the ability tocustom tailor the expression and/or regulatory elements to the host andapplication to achieve a desired expression level that is controlled bythe user. However, endogenous expression also can be utilized in otherembodiments such as by removing a negative regulatory effector orinduction of the gene's promoter when linked to an inducible promoter orother regulatory element. Thus, an endogenous gene having a naturallyoccurring inducible promoter can be up-regulated by providing theappropriate inducing agent, or the regulatory region of an endogenousgene can be engineered to incorporate an inducible regulatory element,thereby allowing the regulation of increased expression of an endogenousgene at a desired time. Similarly, an inducible promoter can be includedas a regulatory element for an exogenous gene introduced into anon-naturally occurring microbial organism.

It is understood that, in methods of the invention, any of the one ormore exogenous nucleic acids can be introduced into a microbial organismto produce a non-naturally occurring microbial organism of theinvention. The nucleic acids can be introduced so as to confer, forexample, a p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate biosynthetic pathway ontothe microbial organism. Alternatively, encoding nucleic acids can beintroduced to produce an intermediate microbial organism having thebiosynthetic capability to catalyze some of the required reactions toconfer p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate biosynthetic capability. Forexample, a non-naturally occurring microbial organism having ap-toluate, terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonatebiosynthetic pathway can comprise at least two exogenous nucleic acidsencoding desired enzymes or proteins. For example, in a(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway, a combination ofthe enzymes expressed can be a combination of2-C-methyl-D-erythritol-4-phosphate dehydratase and1-deoxyxylulose-5-phosphate synthase, or2-C-methyl-D-erythritol-4-phosphate dehydratase and1-deoxy-D-xylulose-5-phosphate reductoisomerase. In a p-toluate pathway,a combination of the enzymes expressed can be a combination of2-dehydro-3-deoxyphosphoheptonate synthase and 3-dehydroquinatedehydratase; shikimate kinase and3-phosphoshikimate-2-carboxyvinyltransferase; shikimate kinase andshikimate dehydrogenase and, and the like. Similarly, in a terephthalatepathway, a combination of the expressed enzymes can be p-toluatemethyl-monooxygenase reductase and 4-carboxybenzyl alcoholdehydrogenase; or 4-carboxybenzyl alcohol dehydrogenase and4-carboxybenzyl aldehyde dehydrogenase, and the like. Thus, it isunderstood that any combination of two or more enzymes or proteins of abiosynthetic pathway can be included in a non-naturally occurringmicrobial organism of the invention. Similarly, it is understood thatany combination of three or more enzymes or proteins of a biosyntheticpathway can be included in a non-naturally occurring microbial organismof the invention, for example, 3-dehydroquinate synthase, shikimatedehydrogenase and shikimate kinase; shikimate kinase, chorismatesynthase and chorismate lyase; 3-dehydroquinate dehydratase, chorismatesynthase and chorismate lyase, and so forth, as desired, so long as thecombination of enzymes and/or proteins of the desired biosyntheticpathway results in production of the corresponding desired product.Similarly, any combination of four, five, six, seven or more enzymes orproteins of a biosynthetic pathway, depending on the pathway asdisclosed herein, can be included in a non-naturally occurring microbialorganism of the invention, as desired, so long as the combination ofenzymes and/or proteins of the desired biosynthetic pathway results inproduction of the corresponding desired product.

In addition to the biosynthesis of p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate as described herein, thenon-naturally occurring microbial organisms and methods of the inventionalso can be utilized in various combinations with each other and withother microbial organisms and methods well known in the art to achieveproduct biosynthesis by other routes. For example, one alternative toproduce p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate other than use of thep-toluate, terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonateproducers is through addition of another microbial organism capable ofconverting a p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway intermediate top-toluate, terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate.One such procedure includes, for example, the fermentation of amicrobial organism that produces a p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway intermediate. Thep-toluate, terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonatepathway intermediate can then be used as a substrate for a secondmicrobial organism that converts the p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway intermediate top-toluate, terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate.The p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway intermediate can beadded directly to another culture of the second organism or the originalculture of the p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway intermediateproducers can be depleted of these microbial organisms by, for example,cell separation, and then subsequent addition of the second organism tothe fermentation broth can be utilized to produce the final productwithout intermediate purification steps.

In other embodiments, the non-naturally occurring microbial organismsand methods of the invention can be assembled in a wide variety ofsubpathways to achieve biosynthesis of, for example, p-toluate,terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate. In theseembodiments, biosynthetic pathways for a desired product of theinvention can be segregated into different microbial organisms, and thedifferent microbial organisms can be co-cultured to produce the finalproduct. In such a biosynthetic scheme, the product of one microbialorganism is the substrate for a second microbial organism until thefinal product is synthesized. For example, the biosynthesis ofp-toluate, terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonatecan be accomplished by constructing a microbial organism that containsbiosynthetic pathways for conversion of one pathway intermediate toanother pathway intermediate or the product. Alternatively, p-toluate,terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate also can bebiosynthetically produced from microbial organisms through co-culture orco-fermentation using two organisms in the same vessel, where the firstmicrobial organism produces a p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate intermediate and the secondmicrobial organism converts the intermediate to p-toluate, terephthalateor (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate.

Given the teachings and guidance provided herein, those skilled in theart will understand that a wide variety of combinations and permutationsexist for the non-naturally occurring microbial organisms and methods ofthe invention together with other microbial organisms, with theco-culture of other non-naturally occurring microbial organisms havingsubpathways and with combinations of other chemical and/or biochemicalprocedures well known in the art to produce p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate.

Sources of encoding nucleic acids for a p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway enzyme or proteincan include, for example, any species where the encoded gene product iscapable of catalyzing the referenced reaction. Such species include bothprokaryotic and eukaryotic organisms including, but not limited to,bacteria, including archaea and eubacteria, and eukaryotes, includingyeast, plant, insect, animal, and mammal, including human. Exemplaryspecies for such sources include, for example, Escherichia coli,Mycobacterium tuberculosis, Agrobacterium tumefaciens, Bacillussubtilis, Synechocystis species, Arabidopsis thaliana, Zymomonasmobilis, Klebsiella oxytoca, Salmonella typhimurium, Salmonella typhi,Lactobacullus collinoides, Klebsiella pneumoniae, Clostridiumpasteuranum, Citrobacter freundii, Clostridium butyricum, Roseburiainulinivorans, Sulfolobus solfataricus, Neurospora crassa, Sinorhizobiumfredii, Helicobacter pylori, Pyrococcus furiosus, Haemophilusinfluenzae, Erwinia chlysanthemi, Staphylococcus aureus, Dunaliellasaliva, Streptococcus pneumoniae, Saccharomyces cerevisiae, Aspergillusnidulans, Pneumocystis carinii, Streptomyces coelicolor, species fromthe genera Burkholderia, Alcaligenes, Pseudomonas, Shingomonas andComamonas, for example, Comamonas testosteroni, as well as otherexemplary species disclosed herein or available as source organisms forcorresponding genes. However, with the complete genome sequenceavailable for now more than 550 species (with more than half of theseavailable on public databases such as the NCBI), including 395microorganism genomes and a variety of yeast, fungi, plant, andmammalian genomes, the identification of genes encoding the requisitep-toluate, terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonatebiosynthetic activity for one or more genes in related or distantspecies, including for example, homologues, orthologs, paralogs andnonorthologous gene displacements of known genes, and the interchange ofgenetic alterations between organisms is routine and well known in theart. Accordingly, the metabolic alterations allowing biosynthesis ofp-toluate, terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonatedescribed herein with reference to a particular organism such as E. colican be readily applied to other microorganisms, including prokaryoticand eukaryotic organisms alike. Given the teachings and guidanceprovided herein, those skilled in the art will know that a metabolicalteration exemplified in one organism can be applied equally to otherorganisms.

In some instances, such as when an alternative p-toluate, terephthalateor (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate biosynthetic pathwayexists in an unrelated species, p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate biosynthesis can beconferred onto the host species by, for example, exogenous expression ofa paralog or paralogs from the unrelated species that catalyzes asimilar, yet non-identical metabolic reaction to replace the referencedreaction. Because certain differences among metabolic networks existbetween different organisms, those skilled in the art will understandthat the actual gene usage between different organisms may differ.However, given the teachings and guidance provided herein, those skilledin the art also will understand that the teachings and methods of theinvention can be applied to all microbial organisms using the cognatemetabolic alterations to those exemplified herein to construct amicrobial organism in a species of interest that will synthesizep-toluate, terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate.

Methods for constructing and testing the expression levels of anon-naturally occurring p-toluate-, terephthalate- or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate-producing host can beperformed, for example, by recombinant and detection methods well knownin the art. Such methods can be found described in, for example,Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., ColdSpring Harbor Laboratory, New York (2001); and Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley and Sons, Baltimore, Md.(1999).

Exogenous nucleic acid sequences involved in a pathway for production ofp-toluate, terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonatecan be introduced stably or transiently into a host cell usingtechniques well known in the art including, but not limited to,conjugation, electroporation, chemical transformation, transduction,transfection, and ultrasound transformation. For exogenous expression inE. coli or other prokaryotic cells, some nucleic acid sequences in thegenes or cDNAs of eukaryotic nucleic acids can encode targeting signalssuch as an N-terminal mitochondrial or other targeting signal, which canbe removed before transformation into prokaryotic host cells, ifdesired. For example, removal of a mitochondrial leader sequence led toincreased expression in E. coli (Hoffmeister et al., J. Biol. Chem.280:4329-4338 (2005)). For exogenous expression in yeast or othereukaryotic cells, genes can be expressed in the cytosol without theaddition of leader sequence, or can be targeted to mitochondrion orother organelles, or targeted for secretion, by the addition of asuitable targeting sequence such as a mitochondrial targeting orsecretion signal suitable for the host cells. Thus, it is understoodthat appropriate modifications to a nucleic acid sequence to remove orinclude a targeting sequence can be incorporated into an exogenousnucleic acid sequence to impart desirable properties. Furthermore, genescan be subjected to codon optimization with techniques well known in theart to achieve optimized expression of the proteins.

An expression vector or vectors can be constructed to include one ormore p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate biosynthetic pathwayencoding nucleic acids as exemplified herein operably linked toexpression control sequences functional in the host organism. Expressionvectors applicable for use in the microbial host organisms of theinvention include, for example, plasmids, phage vectors, viral vectors,episomes and artificial chromosomes, including vectors and selectionsequences or markers operable for stable integration into a hostchromosome. Additionally, the expression vectors can include one or moreselectable marker genes and appropriate expression control sequences.Selectable marker genes also can be included that, for example, provideresistance to antibiotics or toxins, complement auxotrophicdeficiencies, or supply critical nutrients not in the culture media.Expression control sequences can include constitutive and induciblepromoters, transcription enhancers, transcription terminators, and thelike which are well known in the art. When two or more exogenousencoding nucleic acids are to be co-expressed, both nucleic acids can beinserted, for example, into a single expression vector or in separateexpression vectors. For single vector expression, the encoding nucleicacids can be operationally linked to one common expression controlsequence or linked to different expression control sequences, such asone inducible promoter and one constitutive promoter. The transformationof exogenous nucleic acid sequences involved in a metabolic or syntheticpathway can be confirmed using methods well known in the art. Suchmethods include, for example, nucleic acid analysis such as Northernblots or polymerase chain reaction (PCR) amplification of mRNA, orimmunoblotting for expression of gene products, or other suitableanalytical methods to test the expression of an introduced nucleic acidsequence or its corresponding gene product. It is understood by thoseskilled in the art that the exogenous nucleic acid is expressed in asufficient amount to produce the desired product, and it is furtherunderstood that expression levels can be optimized to obtain sufficientexpression using methods well known in the art and as disclosed herein.

The invention additionally provides a method for producing(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, comprising culturing thenon-naturally occurring microbial organism containing a(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway under conditions andfor a sufficient period of time to produce(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate. Such a microbial organismcan have a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathwaycomprising at least one exogenous nucleic acid encoding a(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway enzyme expressed ina sufficient amount to produce(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, the(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway comprising2-C-methyl-D-erythritol-4-phosphate dehydratase (see Example I and FIG.1, step C). A (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway canoptionally further comprise 1-deoxyxylulose-5-phosphate synthase and/or1-deoxy-D-xylulose-5-phosphate reductoisomerase (see Example I and FIG.1, steps A and B).

In another embodiment, the invention provides a method for producingp-toluate, comprising culturing the non-naturally occurring microbialorganism comprising a p-toluate pathway under conditions and for asufficient period of time to produce p-toluate. A p-toluate pathway cancomprise at least one exogenous nucleic acid encoding a p-toluatepathway enzyme expressed in a sufficient amount to produce p-toluate,the p-toluate pathway comprising 2-dehydro-3-deoxyphosphoheptonatesynthase; 3-dehydroquinate synthase; 3-dehydroquinate dehydratase;shikimate dehydrogenase; shikimate kinase;3-phosphoshikimate-2-carboxyvinyltransferase; chorismate synthase;and/or chorismate lyase (see Example II and FIG. 2, steps A-H). Inanother embodiment, a method of the invention can utilize anon-naturally occurring microbial organism that further comprises a(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway (see Example I andFIG. 1). Such a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway cancomprise 2-C-methyl-D-erythritol-4-phosphate dehydratase,1-deoxyxylulose-5-phosphate synthase and/or1-deoxy-D-xylulose-5-phosphate reductoisomerase (see Example I and FIG.1).

The invention further provides a method for producing terephthalate,comprising culturing a non-naturally occurring microbial organismcontaining a terephthalate pathway under conditions and for a sufficientperiod of time to produce terephthalate. Such a terephthalate pathwaycan comprise at least one exogenous nucleic acid encoding aterephthalate pathway enzyme expressed in a sufficient amount to produceterephthalate, the terephthalate pathway comprising p-toluatemethyl-monooxygenase reductase; 4-carboxybenzyl alcohol dehydrogenase;and/or 4-carboxybenzyl aldehyde dehydrogenase. Such a microbial organismcan further comprise a p-toluate pathway, wherein the p-toluate pathwaycomprises 2-dehydro-3-deoxyphosphoheptonate synthase; 3-dehydroquinatesynthase; 3-dehydroquinate dehydratase; shikimate dehydrogenase;shikimate kinase; 3-phosphoshikimate-2-carboxyvinyltransferase;chorismate synthase; and/or chorismate lyase (see Examples 2 and 3 andFIGS. 2 and 3). In another embodiment, the non-naturally occurringmicrobial organism can further comprise a(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway (see Example I andFIG. 1). Thus, in a particular embodiment, the invention provides anon-naturally occurring microbial organism and methods of use, in whichthe microbial organism contains p-toluate, terephthalate and(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathways.

Suitable purification and/or assays to test for the production ofp-toluate, terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonatecan be performed using well known methods. Suitable replicates such astriplicate cultures can be grown for each engineered strain to betested. For example, product and byproduct formation in the engineeredproduction host can be monitored. The final product and intermediates,and other organic compounds, can be analyzed by methods such as HPLC(High Performance Liquid Chromatography), GC-MS (Gas Chromatography-MassSpectroscopy), LC-MS (Liquid Chromatography-Mass Spectroscopy), andUV-visible spectroscopy or other suitable analytical methods usingroutine procedures well known in the art. The release of product in thefermentation broth can also be tested with the culture supernatant.Byproducts and residual glucose can be quantified by HPLC using, forexample, a refractive index detector for glucose and alcohols, and a UVdetector for organic acids (Lin et al., Biotechnol. Bioeng. 90:775-779(2005)), or other suitable assay and detection methods well known in theart. The individual enzyme or protein activities from the exogenous DNAsequences can also be assayed using methods well known in the art. Forexample, p-toluate methyl-monooxygenase activity can be assayed byincubating purified enzyme with NADH, FeSO₄ and the p-toluate substratein a water bath, stopping the reaction by precipitation of the proteins,and analysis of the products in the supernatant by HPLC (Locher et al.,J. Bacteriol. 173:3741-3748 (1991)).

The p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate can be separated from othercomponents in the culture using a variety of methods well known in theart. Such separation methods include, for example, extraction proceduresas well as methods that include continuous liquid-liquid extraction,pervaporation, membrane filtration, membrane separation, reverseosmosis, electrodialysis, distillation, crystallization, centrifugation,extractive filtration, ion exchange chromatography, size exclusionchromatography, adsorption chromatography, and ultrafiltration. All ofthe above methods are well known in the art.

Any of the non-naturally occurring microbial organisms described hereincan be cultured to produce and/or secrete the biosynthetic products ofthe invention. For example, the p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate producers can be culturedfor the biosynthetic production of p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate.

For the production of p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, the recombinant strains arecultured in a medium with carbon source and other essential nutrients.It is sometimes desirable to maintain anaerobic conditions in thefermenter to reduce the cost of the overall process. Such conditions canbe obtained, for example, by first sparging the medium with nitrogen andthen sealing the flasks with a septum and crimp-cap. For strains wheregrowth is not observed anaerobically, microaerobic conditions can beapplied by perforating the septum with a small hole for limitedaeration. Exemplary anaerobic conditions have been described previouslyand are well-known in the art. Exemplary aerobic and anaerobicconditions are described, for example, in United State publication2009/0047719, filed Aug. 10, 2007. Fermentations can be performed in abatch, fed-batch or continuous manner, as disclosed herein.

If desired, the pH of the medium can be maintained at a desired pH, inparticular neutral pH, such as a pH of around 7 by addition of a base,such as NaOH or other bases, or acid, as needed to maintain the culturemedium at a desirable pH. The growth rate can be determined by measuringoptical density using a spectrophotometer (600 nm), and the glucoseuptake rate by monitoring carbon source depletion over time.

The growth medium can include, for example, any carbohydrate sourcewhich can supply a source of carbon to the non-naturally occurringmicroorganism. Such sources include, for example, sugars such asglucose, xylose, arabinose, galactose, mannose, fructose, sucrose andstarch. Other sources of carbohydrate include, for example, renewablefeedstocks and biomass. Exemplary types of biomasses that can be used asfeedstocks in the methods of the invention include cellulosic biomass,hemicellulosic biomass and lignin feedstocks or portions of feedstocks.Such biomass feedstocks contain, for example, carbohydrate substratesuseful as carbon sources such as glucose, xylose, arabinose, galactose,mannose, fructose and starch. Given the teachings and guidance providedherein, those skilled in the art will understand that renewablefeedstocks and biomass other than those exemplified above also can beused for culturing the microbial organisms of the invention for theproduction of p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate.

In addition to renewable feedstocks such as those exemplified above, thep-toluate, terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonatemicrobial organisms of the invention also can be modified for growth onsyngas as its source of carbon. In this specific embodiment, one or moreproteins or enzymes are expressed in the p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate producing organisms toprovide a metabolic pathway for utilization of syngas or other gaseouscarbon source.

Synthesis gas, also known as syngas or producer gas, is the majorproduct of gasification of coal and of carbonaceous materials such asbiomass materials, including agricultural crops and residues. Syngas isa mixture primarily of H₂ and CO and can be obtained from thegasification of any organic feedstock, including but not limited tocoal, coal oil, natural gas, biomass, and waste organic matter.Gasification is generally carried out under a high fuel to oxygen ratio.Although largely H₂ and CO, syngas can also include CO₂ and other gasesin smaller quantities. Thus, synthesis gas provides a cost effectivesource of gaseous carbon such as CO and, additionally, CO₂.

The Wood-Ljungdahl pathway catalyzes the conversion of CO and H₂ toacetyl-CoA and other products such as acetate. Organisms capable ofutilizing CO and syngas also generally have the capability of utilizingCO₂ and CO₂/H₂ mixtures through the same basic set of enzymes andtransformations encompassed by the Wood-Ljungdahl pathway. H₂-dependentconversion of CO₂ to acetate by microorganisms was recognized longbefore it was revealed that CO also could be used by the same organismsand that the same pathways were involved. Many acetogens have been shownto grow in the presence of CO₂ and produce compounds such as acetate aslong as hydrogen is present to supply the necessary reducing equivalents(see for example, Drake, Acetogenesis, pp. 3-60 Chapman and Hall, NewYork, (1994)). This can be summarized by the following equation:

2CO₂+4H₂+n ADP+n Pi→CH₃COOH+2H₂O+n ATP

Hence, non-naturally occurring microorganisms possessing theWood-Ljungdahl pathway can utilize CO₂ and H₂ mixtures as well for theproduction of acetyl-CoA and other desired products.

The Wood-Ljungdahl pathway is well known in the art and consists of 12reactions which can be separated into two branches: (1) methyl branchand (2) carbonyl branch. The methyl branch converts syngas tomethyl-tetrahydrofolate (methyl-THF) whereas the carbonyl branchconverts methyl-THF to acetyl-CoA. The reactions in the methyl branchare catalyzed in order by the following enzymes or proteins: ferredoxinoxidoreductase, formate dehydrogenase, formyltetrahydrofolatesynthetase, methenyltetrahydrofolate cyclodehydratase,methylenetetrahydrofolate dehydrogenase and methylenetetrahydrofolatereductase. The reactions in the carbonyl branch are catalyzed in orderby the following enzymes or proteins: methyltetrahydrofolate:corrinoidprotein methyltransferase (for example, AcsE), corrinoid iron-sulfurprotein, nickel-protein assembly protein (for example, AcsF),ferredoxin, acetyl-CoA synthase, carbon monoxide dehydrogenase andnickel-protein assembly protein (for example, CooC). Following theteachings and guidance provided herein for introducing a sufficientnumber of encoding nucleic acids to generate a p-toluate, terephthalateor (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway, those skilled inthe art will understand that the same engineering design also can beperformed with respect to introducing at least the nucleic acidsencoding the Wood-Ljungdahl enzymes or proteins absent in the hostorganism. Therefore, introduction of one or more encoding nucleic acidsinto the microbial organisms of the invention such that the modifiedorganism contains the complete Wood-Ljungdahl pathway will confer syngasutilization ability.

The reductive tricarboxylic acid cycle coupled with carbon monoxidedehydrogenase and/or hydrogenase activities can also allow theconversion of CO, CO₂ and/or H₂ to acetyl-CoA and other products such asacetate. Organisms capable of fixing carbon via the reductive TCApathway can utilize one or more of the following enzymes: ATPcitrate-lyase, citrate lyase, aconitase, isocitrate dehydrogenase,alpha-ketoglutarate:ferredoxin oxidoreductase, succinyl-CoA synthetase,succinyl-CoA transferase, fumarate reductase, fumarase, malatedehydrogenase, NAD(P)H:ferredoxin oxidoreductase, carbon monoxidedehydrogenase, and hydrogenase. Specifically, the reducing equivalentsextracted from CO and/or H₂ by carbon monoxide dehydrogenase andhydrogenase are utilized to fix CO₂ via the reductive TCA cycle intoacetyl-CoA or acetate. Acetate can be converted to acetyl-CoA by enzymessuch as acetyl-CoA transferase, acetate kinase/phosphotransacetylase,and acetyl-CoA synthetase. Acetyl-CoA can be converted to the p-toluate,terepathalate, or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonateprecursors, glyceraldehyde-3-phosphate, phosphoenolpyruvate, andpyruvate, by pyruvate:ferredoxin oxidoreductase and the enzymes ofgluconeogenesis. Following the teachings and guidance provided hereinfor introducing a sufficient number of encoding nucleic acids togenerate a p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway, those skilled inthe art will understand that a similar engineering design also can beperformed with respect to introducing at least the nucleic acidsencoding the reductive TCA pathway enzymes or proteins absent in thehost organism. Therefore, introduction of one or more encoding nucleicacids into the microbial organisms of the invention such that themodified organism contains the complete reductive TCA pathway willconfer syngas utilization ability.

Accordingly, given the teachings and guidance provided herein, thoseskilled in the art will understand that a non-naturally occurringmicrobial organism can be produced that secretes the biosynthesizedcompounds of the invention when grown on a carbon source such as acarbohydrate. Such compounds include, for example, p-toluate,terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate and any ofthe intermediate metabolites in the p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway. All that isrequired is to engineer in one or more of the required enzyme or proteinactivities to achieve biosynthesis of the desired compound orintermediate including, for example, inclusion of some or all of thep-toluate, terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonatebiosynthetic pathways. Accordingly, the invention provides anon-naturally occurring microbial organism that produces and/or secretesp-toluate, terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonatewhen grown on a carbohydrate or other carbon source and produces and/orsecretes any of the intermediate metabolites shown in the p-toluate,terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathwaywhen grown on a carbohydrate or other carbon source. The p-toluate,terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate producingmicrobial organisms of the invention can initiate synthesis from anintermediate. For example, a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonatepathway intermediate can be 1-deoxy-D-xylulose-5-phosphate orC-methyl-D-erythritol-4-phosphate (see Example I and FIG. 1). Ap-toluate pathway intermediate can be, for example,2,4-dihydroxy-5-methyl-6-[(phosphonooxy)methyl]oxane-2-carboxylate,1,3-dihydroxy-4-methyl-5-oxocyclohexane-1-carboxylate,5-hydroxy-4-methyl-3-oxocyclohex-1-ene-1-carboxylate,3,5-dihydroxy-4-methylcyclohex-1-ene-1-carboxylate,5-hydroxy-4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate,5-[(1-carboxyeth-1-en-1-yl)oxy]-4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate,or3-[(1-carboxyeth-1-en-1-yl)oxy]-4-methylcyclohexa-1,5-diene-1-carboxylate(see Example II and FIG. 2). A terephthalate intermediate can be, forexample, 4-carboxybenzyl alcohol or 4-carboxybenzaldehyde (see ExampleIII and FIG. 3).

The non-naturally occurring microbial organisms of the invention areconstructed using methods well known in the art as exemplified herein toexogenously express at least one nucleic acid encoding a p-toluate,terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathwayenzyme or protein in sufficient amounts to produce p-toluate,terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate. It isunderstood that the microbial organisms of the invention are culturedunder conditions sufficient to produce p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate. Following the teachings andguidance provided herein, the non-naturally occurring microbialorganisms of the invention can achieve biosynthesis of p-toluate,terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate resultingin intracellular concentrations between about 0.1-200 mM or more.Generally, the intracellular concentration of p-toluate, terephthalateor (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate is between about 3-150mM, particularly between about 5-125 mM and more particularly betweenabout 8-100 mM, including about 10 mM, 20 mM, 50 mM, 80 mM, or more.Intracellular concentrations between and above each of these exemplaryranges also can be achieved from the non-naturally occurring microbialorganisms of the invention.

In some embodiments, culture conditions include anaerobic orsubstantially anaerobic growth or maintenance conditions. Exemplaryanaerobic conditions have been described previously and are well knownin the art. Exemplary anaerobic conditions for fermentation processesare described herein and are described, for example, in U.S. publication2009/0047719, filed Aug. 10, 2007. Any of these conditions can beemployed with the non-naturally occurring microbial organisms as well asother anaerobic conditions well known in the art. The p-toluate,terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate producerscan synthesize p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate at intracellularconcentrations of 5-10 mM or more as well as all other concentrationsexemplified herein under substantially anaerobic conditions. It isunderstood that, even though the above description refers tointracellular concentrations, p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate producing microbialorganisms can produce p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate intracellularly and/orsecrete the product into the culture medium.

In addition to the culturing and fermentation conditions disclosedherein, growth conditions for achieving biosynthesis of p-toluate,terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate can includethe addition of an osmoprotectant to the culturing conditions. Incertain embodiments, the non-naturally occurring microbial organisms ofthe invention can be sustained, cultured or fermented as describedherein in the presence of an osmoprotectant. Briefly, an osmoprotectantrefers to a compound that acts as an osmolyte and helps a microbialorganism as described herein survive osmotic stress. Osmoprotectantsinclude, but are not limited to, betaines, amino acids, and the sugartrehalose. Non-limiting examples of such are glycine betaine, pralinebetaine, dimethylthetin, dimethylslfonioproprionate,3-dimethylsulfonio-2-methylproprionate, pipecolic acid,dimethylsulfonioacetate, choline, L-carnitine and ectoine. In oneaspect, the osmoprotectant is glycine betaine. It is understood to oneof ordinary skill in the art that the amount and type of osmoprotectantsuitable for protecting a microbial organism described herein fromosmotic stress will depend on the microbial organism used. The amount ofosmoprotectant in the culturing conditions can be, for example, no morethan about 0.1 mM, no more than about 0.5 mM, no more than about 1.0 mM,no more than about 1.5 mM, no more than about 2.0 mM, no more than about2.5 mM, no more than about 3.0 mM, no more than about 5.0 mM, no morethan about 7.0 mM, no more than about 10 mM, no more than about 50 mM,no more than about 100 mM or no more than about 500 mM.

The culture conditions can include, for example, liquid cultureprocedures as well as fermentation and other large scale cultureprocedures. As described herein, particularly useful yields of thebiosynthetic products of the invention can be obtained under anaerobicor substantially anaerobic culture conditions.

As described herein, one exemplary growth condition for achievingbiosynthesis of p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate includes anaerobic cultureor fermentation conditions. In certain embodiments, the non-naturallyoccurring microbial organisms of the invention can be sustained,cultured or fermented under anaerobic or substantially anaerobicconditions. Briefly, anaerobic conditions refers to an environmentdevoid of oxygen. Substantially anaerobic conditions include, forexample, a culture, batch fermentation or continuous fermentation suchthat the dissolved oxygen concentration in the medium remains between 0and 10% of saturation. Substantially anaerobic conditions also includesgrowing or resting cells in liquid medium or on solid agar inside asealed chamber maintained with an atmosphere of less than 1% oxygen. Thepercent of oxygen can be maintained by, for example, sparging theculture with an N₂/CO₂ mixture or other suitable non-oxygen gas orgases.

The culture conditions described herein can be scaled up and growncontinuously for manufacturing of p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate. Exemplary growth proceduresinclude, for example, fed-batch fermentation and batch separation;fed-batch fermentation and continuous separation, or continuousfermentation and continuous separation. All of these processes are wellknown in the art. Fermentation procedures are particularly useful forthe biosynthetic production of commercial quantities of p-toluate,terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate. Generally,and as with non-continuous culture procedures, the continuous and/ornear-continuous production of p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate will include culturing anon-naturally occurring p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate producing organism of theinvention in sufficient nutrients and medium to sustain and/or nearlysustain growth in an exponential phase. Continuous culture under suchconditions can be include, for example, growth for 1 day, 2, 3, 4, 5, 6or 7 days or more. Additionally, continuous culture can include longertime periods of 1 week, 2, 3, 4 or 5 or more weeks and up to severalmonths. Alternatively, organisms of the invention can be cultured forhours, if suitable for a particular application. It is to be understoodthat the continuous and/or near-continuous culture conditions also caninclude all time intervals in between these exemplary periods. It isfurther understood that the time of culturing the microbial organism ofthe invention is for a sufficient period of time to produce a sufficientamount of product for a desired purpose.

Fermentation procedures are well known in the art. Briefly, fermentationfor the biosynthetic production of p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate can be utilized in, forexample, fed-batch fermentation and batch separation; fed-batchfermentation and continuous separation, or continuous fermentation andcontinuous separation. Examples of batch and continuous fermentationprocedures are well known in the art.

In addition to the above fermentation procedures using the p-toluate,terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate producersof the invention for continuous production of substantial quantities ofp-toluate, terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,the p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate producers also can be, forexample, simultaneously subjected to chemical synthesis procedures toconvert the product to other compounds or the product can be separatedfrom the fermentation culture and sequentially subjected to chemicalconversion to convert the product to other compounds, if desired.

To generate better producers, metabolic modeling can be utilized tooptimize growth conditions. Modeling can also be used to design geneknockouts that additionally optimize utilization of the pathway (see,for example, U.S. patent publications US 2002/0012939, US 2003/0224363,US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 andUS 2004/0009466, and U.S. Pat. No. 7,127,379). Modeling analysis allowsreliable predictions of the effects on cell growth of shifting themetabolism towards more efficient production of p-toluate, terephthalateor (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate.

One computational method for identifying and designing metabolicalterations favoring biosynthesis of a desired product is the OptKnockcomputational framework (Burgard et al., Biotechnol. Bioeng. 84:647-657(2003)). OptKnock is a metabolic modeling and simulation program thatsuggests gene deletion or disruption strategies that result ingenetically stable microorganisms which overproduce the target product.Specifically, the framework examines the complete metabolic and/orbiochemical network of a microorganism in order to suggest geneticmanipulations that force the desired biochemical to become an obligatorybyproduct of cell growth. By coupling biochemical production with cellgrowth through strategically placed gene deletions or other functionalgene disruption, the growth selection pressures imposed on theengineered strains after long periods of time in a bioreactor lead toimprovements in performance as a result of the compulsory growth-coupledbiochemical production. Lastly, when gene deletions are constructedthere is a negligible possibility of the designed strains reverting totheir wild-type states because the genes selected by OptKnock are to becompletely removed from the genome. Therefore, this computationalmethodology can be used to either identify alternative pathways thatlead to biosynthesis of a desired product or used in connection with thenon-naturally occurring microbial organisms for further optimization ofbiosynthesis of a desired product.

Briefly, OptKnock is a term used herein to refer to a computationalmethod and system for modeling cellular metabolism. The OptKnock programrelates to a framework of models and methods that incorporate particularconstraints into flux balance analysis (FBA) models. These constraintsinclude, for example, qualitative kinetic information, qualitativeregulatory information, and/or DNA microarray experimental data.OptKnock also computes solutions to various metabolic problems by, forexample, tightening the flux boundaries derived through flux balancemodels and subsequently probing the performance limits of metabolicnetworks in the presence of gene additions or deletions. OptKnockcomputational framework allows the construction of model formulationsthat allow an effective query of the performance limits of metabolicnetworks and provides methods for solving the resulting mixed-integerlinear programming problems. The metabolic modeling and simulationmethods referred to herein as OptKnock are described in, for example,U.S. publication 2002/0168654, filed Jan. 10, 2002, in InternationalPatent No. PCT/US02/00660, filed Jan. 10, 2002, and U.S. publication2009/0047719, filed Aug. 10, 2007.

Another computational method for identifying and designing metabolicalterations favoring biosynthetic production of a product is a metabolicmodeling and simulation system termed SimPheny®. This computationalmethod and system is described in, for example, U.S. publication2003/0233218, filed Jun. 14, 2002, and in International PatentApplication No. PCT/US03/18838, filed Jun. 13, 2003. SimPheny® is acomputational system that can be used to produce a network model insilico and to simulate the flux of mass, energy or charge through thechemical reactions of a biological system to define a solution spacethat contains any and all possible functionalities of the chemicalreactions in the system, thereby determining a range of allowedactivities for the biological system. This approach is referred to asconstraints-based modeling because the solution space is defined byconstraints such as the known stoichiometry of the included reactions aswell as reaction thermodynamic and capacity constraints associated withmaximum fluxes through reactions. The space defined by these constraintscan be interrogated to determine the phenotypic capabilities andbehavior of the biological system or of its biochemical components.

These computational approaches are consistent with biological realitiesbecause biological systems are flexible and can reach the same result inmany different ways. Biological systems are designed throughevolutionary mechanisms that have been restricted by fundamentalconstraints that all living systems must face. Therefore,constraints-based modeling strategy embraces these general realities.Further, the ability to continuously impose further restrictions on anetwork model via the tightening of constraints results in a reductionin the size of the solution space, thereby enhancing the precision withwhich physiological performance or phenotype can be predicted.

Given the teachings and guidance provided herein, those skilled in theart will be able to apply various computational frameworks for metabolicmodeling and simulation to design and implement biosynthesis of adesired compound in host microbial organisms. Such metabolic modelingand simulation methods include, for example, the computational systemsexemplified above as SimPheny® and OptKnock. For illustration of theinvention, some methods are described herein with reference to theOptKnock computation framework for modeling and simulation. Thoseskilled in the art will know how to apply the identification, design andimplementation of the metabolic alterations using OptKnock to any ofsuch other metabolic modeling and simulation computational frameworksand methods well known in the art.

The methods described above will provide one set of metabolic reactionsto disrupt. Elimination of each reaction within the set or metabolicmodification can result in a desired product as an obligatory productduring the growth phase of the organism. Because the reactions areknown, a solution to the bilevel OptKnock problem also will provide theassociated gene or genes encoding one or more enzymes that catalyze eachreaction within the set of reactions. Identification of a set ofreactions and their corresponding genes encoding the enzymesparticipating in each reaction is generally an automated process,accomplished through correlation of the reactions with a reactiondatabase having a relationship between enzymes and encoding genes.

Once identified, the set of reactions that are to be disrupted in orderto achieve production of a desired product are implemented in the targetcell or organism by functional disruption of at least one gene encodingeach metabolic reaction within the set. One particularly useful means toachieve functional disruption of the reaction set is by deletion of eachencoding gene. However, in some instances, it can be beneficial todisrupt the reaction by other genetic aberrations including, forexample, mutation, deletion of regulatory regions such as promoters orcis binding sites for regulatory factors, or by truncation of the codingsequence at any of a number of locations. These latter aberrations,resulting in less than total deletion of the gene set can be useful, forexample, when rapid assessments of the coupling of a product are desiredor when genetic reversion is less likely to occur.

To identify additional productive solutions to the above describedbilevel OptKnock problem which lead to further sets of reactions todisrupt or metabolic modifications that can result in the biosynthesis,including growth-coupled biosynthesis of a desired product, anoptimization method, termed integer cuts, can be implemented. Thismethod proceeds by iteratively solving the OptKnock problem exemplifiedabove with the incorporation of an additional constraint referred to asan integer cut at each iteration. Integer cut constraints effectivelyprevent the solution procedure from choosing the exact same set ofreactions identified in any previous iteration that obligatorily couplesproduct biosynthesis to growth. For example, if a previously identifiedgrowth-coupled metabolic modification specifies reactions 1, 2, and 3for disruption, then the following constraint prevents the samereactions from being simultaneously considered in subsequent solutions.The integer cut method is well known in the art and can be founddescribed in, for example, Burgard et al., Biotechnol. Prog. 17:791-797(2001). As with all methods described herein with reference to their usein combination with the OptKnock computational framework for metabolicmodeling and simulation, the integer cut method of reducing redundancyin iterative computational analysis also can be applied with othercomputational frameworks well known in the art including, for example,SimPheny®.

The methods exemplified herein allow the construction of cells andorganisms that biosynthetically produce a desired product, including theobligatory coupling of production of a target biochemical product togrowth of the cell or organism engineered to harbor the identifiedgenetic alterations. Therefore, the computational methods describedherein allow the identification and implementation of metabolicmodifications that are identified by an in silico method selected fromOptKnock or SimPheny®. The set of metabolic modifications can include,for example, addition of one or more biosynthetic pathway enzymes and/orfunctional disruption of one or more metabolic reactions including, forexample, disruption by gene deletion.

As discussed above, the OptKnock methodology was developed on thepremise that mutant microbial networks can be evolved towards theircomputationally predicted maximum-growth phenotypes when subjected tolong periods of growth selection. In other words, the approach leveragesan organism's ability to self-optimize under selective pressures. TheOptKnock framework allows for the exhaustive enumeration of genedeletion combinations that force a coupling between biochemicalproduction and cell growth based on network stoichiometry. Theidentification of optimal gene/reaction knockouts requires the solutionof a bilevel optimization problem that chooses the set of activereactions such that an optimal growth solution for the resulting networkoverproduces the biochemical of interest (Burgard et al., Biotechnol.Bioeng. 84:647-657 (2003)).

An in silico stoichiometric model of E. coli metabolism can be employedto identify essential genes for metabolic pathways as exemplifiedpreviously and described in, for example, U.S. patent publications US2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US2003/0059792, US 2002/0168654 and US 2004/0009466, and in U.S. Pat. No.7,127,379. As disclosed herein, the OptKnock mathematical framework canbe applied to pinpoint gene deletions leading to the growth-coupledproduction of a desired product. Further, the solution of the bilevelOptKnock problem provides only one set of deletions. To enumerate allmeaningful solutions, that is, all sets of knockouts leading togrowth-coupled production formation, an optimization technique, termedinteger cuts, can be implemented. This entails iteratively solving theOptKnock problem with the incorporation of an additional constraintreferred to as an integer cut at each iteration, as discussed above.

As disclosed herein, a nucleic acid encoding a desired activity of ap-toluate, terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonatepathway can be introduced into a host organism. In some cases, it can bedesirable to modify an activity of a p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway enzyme or protein toincrease production of p-toluate, terephthalate or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate. For example, knownmutations that increase the activity of a protein or enzyme can beintroduced into an encoding nucleic acid molecule. Additionally,optimization methods can be applied to increase the activity of anenzyme or protein and/or decrease an inhibitory activity, for example,decrease the activity of a negative regulator.

One such optimization method is directed evolution. Directed evolutionis a powerful approach that involves the introduction of mutationstargeted to a specific gene in order to improve and/or alter theproperties of an enzyme. Improved and/or altered enzymes can beidentified through the development and implementation of sensitivehigh-throughput screening assays that allow the automated screening ofmany enzyme variants (for example, >10⁴). Iterative rounds ofmutagenesis and screening typically are performed to afford an enzymewith optimized properties. Computational algorithms that can help toidentify areas of the gene for mutagenesis also have been developed andcan significantly reduce the number of enzyme variants that need to begenerated and screened. Numerous directed evolution technologies havebeen developed (for reviews, see Hibbert et al., Biomol. Eng 22:11-19(2005); Huisman and Lalonde, In Biocatalysis in the pharmaceutical andbiotechnology industries pgs. 717-742 (2007), Patel (ed.), CRC Press;Otten and Quax. Biomol. Eng 22:1-9 (2005).; and Sen et al., ApplBiochem. Biotechnol 143:212-223 (2007)) to be effective at creatingdiverse variant libraries, and these methods have been successfullyapplied to the improvement of a wide range of properties across manyenzyme classes. Enzyme characteristics that have been improved and/oraltered by directed evolution technologies include, for example:selectivity/specificity, for conversion of non-natural substrates;temperature stability, for robust high temperature processing; pHstability, for bioprocessing under lower or higher pH conditions;substrate or product tolerance, so that high product titers can beachieved; binding (K_(m)), including broadening substrate binding toinclude non-natural substrates; inhibition (K_(i)), to remove inhibitionby products, substrates, or key intermediates; activity (kcat), toincreases enzymatic reaction rates to achieve desired flux; expressionlevels, to increase protein yields and overall pathway flux; oxygenstability, for operation of air sensitive enzymes under aerobicconditions; and anaerobic activity, for operation of an aerobic enzymein the absence of oxygen.

A number of exemplary methods have been developed for the mutagenesisand diversification of genes to target desired properties of specificenzymes. Such methods are well known to those skilled in the art. Any ofthese can be used to alter and/or optimize the activity of a p-toluate,terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathwayenzyme or protein. Such methods include, but are not limited to EpPCR,which introduces random point mutations by reducing the fidelity of DNApolymerase in PCR reactions (Pritchard et al., J Theor. Biol.234:497-509 (2005)); Error-prone Rolling Circle Amplification (epRCA),which is similar to epPCR except a whole circular plasmid is used as thetemplate and random 6-mers with exonuclease resistant thiophosphatelinkages on the last 2 nucleotides are used to amplify the plasmidfollowed by transformation into cells in which the plasmid isre-circularized at tandem repeats (Fujii et al., Nucleic Acids Res.32:e145 (2004); and Fujii et al., Nat. Protoc. 1:2493-2497 (2006)); DNAor Family Shuffling, which typically involves digestion of two or morevariant genes with nucleases such as Dnase I or EndoV to generate a poolof random fragments that are reassembled by cycles of annealing andextension in the presence of DNA polymerase to create a library ofchimeric genes (Stemmer, Proc Natl Acad Sci USA 91:10747-10751 (1994);and Stemmer, Nature 370:389-391 (1994)); Staggered Extension (StEP),which entails template priming followed by repeated cycles of 2 step PCRwith denaturation and very short duration of annealing/extension (asshort as 5 sec) (Zhao et al., Nat. Biotechnol. 16:258-261 (1998));Random Priming Recombination (RPR), in which random sequence primers areused to generate many short DNA fragments complementary to differentsegments of the template (Shao et al., Nucleic Acids Res 26:681-683(1998)).

Additional methods include Heteroduplex Recombination, in whichlinearized plasmid DNA is used to form heteroduplexes that are repairedby mismatch repair (Volkov et al, Nucleic Acids Res. 27:el 8 (1999); andVolkov et al., Methods Enzymol. 328:456-463 (2000)); RandomChimeragenesis on Transient Templates (RACHITT), which employs Dnase Ifragmentation and size fractionation of single stranded DNA (ssDNA)(Coco et al., Nat. Biotechnol. 19:354-359 (2001)); Recombined Extensionon Truncated templates (RETT), which entails template switching ofunidirectionally growing strands from primers in the presence ofunidirectional ssDNA fragments used as a pool of templates (Lee et al.,J. Molec. Catalysis 26:119-129 (2003)); Degenerate Oligonucleotide GeneShuffling (DOGS), in which degenerate primers are used to controlrecombination between molecules; (Bergquist and Gibbs, Methods Mol. Biol352:191-204 (2007); Bergquist et al., Biomol. Eng 22:63-72 (2005); Gibbset al., Gene 271:13-20 (2001)); Incremental Truncation for the Creationof Hybrid Enzymes (ITCHY), which creates a combinatorial library with 1base pair deletions of a gene or gene fragment of interest (Ostermeieret al., Proc. Natl. Acad. Sci. USA 96:3562-3567 (1999); and Ostermeieret al., Nat. Biotechnol. 17:1205-1209 (1999)); Thio-IncrementalTruncation for the Creation of Hybrid Enzymes (THIO-ITCHY), which issimilar to ITCHY except that phosphothioate dNTPs are used to generatetruncations (Lutz et al., Nucleic Acids Res 29:E16 (2001)); SCRATCHY,which combines two methods for recombining genes, ITCHY and DNAshuffling (Lutz et al., Proc. Natl. Acad. Sci. USA 98:11248-11253(2001)); Random Drift Mutagenesis (RNDM), in which mutations made viaepPCR are followed by screening/selection for those retaining usableactivity (Bergquist et al., Biomol. Eng. 22:63-72 (2005)); SequenceSaturation Mutagenesis (SeSaM), a random mutagenesis method thatgenerates a pool of random length fragments using random incorporationof a phosphothioate nucleotide and cleavage, which is used as a templateto extend in the presence of “universal” bases such as inosine, andreplication of an inosine-containing complement gives random baseincorporation and, consequently, mutagenesis (Wong et al., Biotechnol.J. 3:74-82 (2008); Wong et al., Nucleic Acids Res. 32:e26 (2004); andWong et al., Anal. Biochem. 341:187-189 (2005)); Synthetic Shuffling,which uses overlapping oligonucleotides designed to encode “all geneticdiversity in targets” and allows a very high diversity for the shuffledprogeny (Ness et al., Nat. Biotechnol. 20:1251-1255 (2002)); NucleotideExchange and Excision Technology NexT, which exploits a combination ofdUTP incorporation followed by treatment with uracil DNA glycosylase andthen piperidine to perform endpoint DNA fragmentation (Muller et al.,Nucleic Acids Res. 33:e117 (2005)).

Further methods include Sequence Homology-Independent ProteinRecombination (SHIPREC), in which a linker is used to facilitate fusionbetween two distantly related or unrelated genes, and a range ofchimeras is generated between the two genes, resulting in libraries ofsingle-crossover hybrids (Sieber et al., Nat. Biotechnol. 19:456-460(2001)); Gene Site Saturation Mutagenesis™ (GSSM™), in which thestarting materials include a supercoiled double stranded DNA (dsDNA)plasmid containing an insert and two primers which are degenerate at thedesired site of mutations (Kretz et al., Methods Enzymol. 388:3-11(2004)); Combinatorial Cassette Mutagenesis (CCM), which involves theuse of short oligonucleotide cassettes to replace limited regions with alarge number of possible amino acid sequence alterations (Reidhaar-Olsonet al. Methods Enzymol. 208:564-586 (1991); and Reidhaar-Olson et al.Science 241:53-57 (1988)); Combinatorial Multiple Cassette Mutagenesis(CMCM), which is essentially similar to CCM and uses epPCR at highmutation rate to identify hot spots and hot regions and then extensionby CMCM to cover a defined region of protein sequence space (Reetz etal., Angew. Chem. Int. Ed Engl. 40:3589-3591 (2001)); the MutatorStrains technique, in which conditional is mutator plasmids, utilizingthe mutD5 gene, which encodes a mutant subunit of DNA polymerase III, toallow increases of 20 to 4000-X in random and natural mutation frequencyduring selection and block accumulation of deleterious mutations whenselection is not required (Selifonova et al., Appl. Environ. Microbiol.67:3645-3649 (2001)); Low et al., J. Mol. Biol. 260:359-3680 (1996)).

Additional exemplary methods include Look-Through Mutagenesis (LTM),which is a multidimensional mutagenesis method that assesses andoptimizes combinatorial mutations of selected amino acids (Rajpal etal., Proc. Natl. Acad. Sci. USA 102:8466-8471 (2005)); Gene Reassembly,which is a DNA shuffling method that can be applied to multiple genes atone time or to create a large library of chimeras (multiple mutations)of a single gene (Tunable GeneReassembly™ (TGR™) Technology supplied byVerenium Corporation), in Silico Protein Design Automation (PDA), whichis an optimization algorithm that anchors the structurally definedprotein backbone possessing a particular fold, and searches sequencespace for amino acid substitutions that can stabilize the fold andoverall protein energetics, and generally works most effectively onproteins with known three-dimensional structures (Hayes et al., Proc.Natl. Acad. Sci. USA 99:15926-15931 (2002)); and Iterative SaturationMutagenesis (ISM), which involves using knowledge of structure/functionto choose a likely site for enzyme improvement, performing saturationmutagenesis at chosen site using a mutagenesis method such as StratageneQuikChange (Stratagene; San Diego Calif.), screening/selecting fordesired properties, and, using improved clone(s), starting over atanother site and continue repeating until a desired activity is achieved(Reetz et al., Nat. Protoc. 2:891-903 (2007); and Reetz et al., Angew.Chem. Int. Ed Engl. 45:7745-7751 (2006)).

Any of the aforementioned methods for mutagenesis can be used alone orin any combination. Additionally, any one or combination of the directedevolution methods can be used in conjunction with adaptive evolutiontechniques, as described herein.

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoprovided within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

EXAMPLE I Exemplary Pathway for Producing(2-Hydroxy-3-methyl-4-oxobutoxy)phosphonate

This example describes an exemplary pathway for producing theterephthalic acid (PTA) precursor(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate (2H3M4OP).

The precursor to the p-toluate and PTA pathways is 2H3M4OP. Thischemical can be derived from central metabolitesglyceraldehyde-3-phosphate (G3P) and pyruvate in three enzymatic stepsas shown in FIG. 1. The first two steps are native to E. coli and otherorganisms that utilize the methylerythritol phosphate (non-mevalonate)pathway for isoprenoid biosynthesis. Pyruvate and G3P are firstcondensed to form 1-deoxy-D-xylulose 5-phosphate (DXP) by DXP synthase.Subsequent reduction and rearrangement of the carbon backbone iscatalyzed by DXP reductoisomerase. Finally, a novel diol dehydratasetransforms 2-C-methyl-D-erythritol-4-phosphate to the p-toluateprecursor 2H3M4OP.

A. 1-Deoxyxylulose-5-phosphate (DXP) synthase. Pyruvate and G3P arecondensed to form DXP by DXP synthase (EC 2.2.1.7). This enzymecatalyzes the first step in the non-mevalonate pathway of isoprenoidbiosynthesis. The enzyme requires thiamine diphosphate as a cofactor,and also requires reduced FAD, although there is no net redox change. Acrystal structure of the E. coli enzyme is available (Xiang et al., J.Biol. Chem. 282:2676-2682 (2007)(doi:M610235200,pii;10.1074/jbc.M610235200 doi). Other enzymes have been cloned andcharacterized in M. tuberculosis (Bailey et al., Glycobiology 12:813-820(2002) and Agrobacterium tumefaciens (Lee et al., J. Biotechnol.128:555-566 (2007)(doi:S0168-1656(06)00966-7,pii;10.1016/j.jbiotec.2006.11.009, doi). DXP synthase enzymes from B.subtilis and Synechocystis sp. PCC 6803 were cloned into E. coli (Harkerand Bramley, FEBS Lett. 448:115-119 (1999)(doi:S0014-5793(99)00360-9,pii).

GenBank Gene Accession No. GI No. Organism dxs AAC73523.1 1786622Escherichia coli dxs P0A554.1 61222979 M. tuberculosis dxs11 AAP56243.137903541 Agrobacterium tumefaciens dxs P54523.1 1731052 Bacillussubtilis sll1945 BAA17089.1 1652165 Synechocystis sp. PCC 6803

B. 1-Deoxy-D-xylulose-5-phosphate reductoisomerase (EC 1.1.1.267). TheNAD(P)H-dependent reduction and rearrangement of1-deoxy-D-xylulose-5-phosphate (DXP) to2-C-methyl-D-erythritol-4-phosphate is catalyzed by DXP reductoisomerase(DXR, EC 1.1.1.267) in the second step of the non-mevalonate pathway forisoprenoid biosynthesis. The NADPH-dependent E. coli enzyme is encodedby dxr (Takahashi et al., Proc. Natl. Acad. Sci. USA 95:9879-9884(1998)). A recombinant enzyme from Arabidopsis thaliana was functionallyexpressed in E. coli (Carretero-Paulet et al., Plant Physiol.129:1581-1591 (2002)(doi:10.1104/pp.003798 (doi). DXR enzymes fromZymomonas mobilis and Mycobacterium tuberculosis have been characterizedand crystal structures are available (Grolle et al., FEMS Microbiol.Lett. 191:131-137 (2000)(doi:S0378-1097(00)00382-7, pii); Henriksson etal., Acta Crystallogr. D. Biol. Crystallogr. 62:807-813(2006)(doi:S0907444906019196, pii;10.1107/S0907444906019196, doi). Mostcharacterized DXR enzymes are strictly NADPH dependent, but the enzymesfrom A. thaliana and M. tuberculosis react with NADH at a reduced rate(Argyrou and Blanchard, Biochemistry 43:4375-4384(2004)(doi:10.1021/bi049974k, doi); Rohdich et al., FEBS J.273:4446-4458 (2006)(doi:EJB5446, pii;10.1111/j.1742-4658.2006.05446.x,doi.

GenBank Gene Accession No. GI No. Organism dxr AAC73284.1 1786369Escherichia coli dxr AAF73140.1 8131928 Arabisopsis thaliana dxrCAB60758.1 6434139 Zymomonas mobilis dxr NP_217386.2 57117032Mycobacterium tuberculosis

C. 2-C-Methyl-D-erythritol-4-phosphate dehydratase. A diol dehydrataseis required to convert 2-C-methyl-D-erythritol-4-phosphate into thep-toluate precursor (Aitmiller and Wagner, Arch. Biochem. Biophys.138:160-170 (1970)). Although this transformation has not beendemonstrated experimentally, several enzymes catalyze similartransformations including dihydroxy-acid dehydratase (EC 4.2.1.9),propanediol dehydratase (EC 4.2.1.28), glycerol dehydratase (EC4.2.1.30) and myo-inositose dehydratase (EC 4.2.1.44).

Diol dehydratase or propanediol dehydratase enzymes (EC 4.2.1.28)capable of converting the secondary diol 2,3-butanediol to 2-butanoneare excellent candidates for this transformation.Adenosylcobalamin-dependent diol dehydratases contain alpha, beta andgamma subunits, which are all required for enzyme function. Exemplarygene candidates are found in Klebsiella pneumoniae (Tobimatsu et al.,Biosci. Biotechnol. Biochem. 62:1774-1777 (1998); Toraya et al.,.Biochem. Biophys. Res. Commun. 69:475-480 (1976)), Salmonellatyphimurium (Bobik et al., J. Bacteriol. 179:6633-6639 (1997)),Klebsiella oxytoca (Tobimatsu et al., J. Biol. Chem. 270:7142-7148(1995)) and Lactobacillus collinoides (Sauvageot et al., FEMS Microbiol.Lett. 209:69-74 (2002)). Methods for isolating diol dehydratase genecandidates in other organisms are well known in the art (see, forexample, U.S. Pat. No. 5,686,276).

GenBank Gene Accession No. GI No. Organism pddA BAA08099.1 868006Klebsiella oxytoca pddB BAA08100.1 868007 Klebsiella oxytoca pddCBAA08101.1 868008 Klebsiella oxytoca pduC AAB84102.1 2587029 Salmonellatyphimurium pduD AAB84103.1 2587030 Salmonella typhimurium pduEAAB84104.1 2587031 Salmonella typhimurium pduC CAC82541.1 18857678Lactobacullus collinoides pduD CAC82542.1 18857679 Lactobaculluscollinoides pduE CAD01091.1 18857680 Lactobacullus collinoides pddAAAC98384.1 4063702 Klebsiella pneumoniae pddB AAC98385.1 4063703Klebsiella pneumoniae pddC AAC98386.1 4063704 Klebsiella pneumoniae

Enzymes in the glycerol dehydratase family (EC 4.2.1.30) can also beused to dehydrate 2-C-methyl-D-erythritol-4-phosphate. Exemplary genecandidates encoded by gldABC and dhaB123 in Klebsiella pneumoniae (WO2008/137403) and (Toraya et al., Biochem. Biophys. Res. Commun.69:475-480 (1976)), dhaBCE in Clostridium pasteuranum (Macis et al., FEMMicrobiol Lett. 164:21-28 (1998)) and dhaBCE in Citrobacter freundii(Seyfried et al., J. Bacteriol. 178:5793-5796 (1996)). Variants of theB12-dependent diol dehydratase from K. pneumoniae with 80- to 336-foldenhanced activity were recently engineered by introducing mutations intwo residues of the beta subunit (Qi et al., J. Biotechnol. 144:43-50(2009)(doi:S0168-1656(09)00258-2, pii;10.1016/j jbiotec.2009.06.015,doi). Diol dehydratase enzymes with reduced inactivation kinetics weredeveloped by DuPont using error-prone PCR (WO 2004/056963).

GenBank Gene Accession No. GI No. Organism gldA AAB96343.1 1778022Klebsiella pneumoniae gldB AAB96344.1 1778023 Klebsiella pneumoniae gldCAAB96345.1 1778024 Klebsiella pneumoniae dhaB1 ABR78884.1 150956854Klebsiella pneumoniae dhaB2 ABR78883.1 150956853 Klebsiella pneumoniaedhaB3 ABR78882.1 150956852 Klebsiella pneumoniae dhaB AAC27922.1 3360389Clostridium pasteuranum dhaC AAC27923.1 3360390 Clostridium pasteuranumdhaE AAC27924.1 3360391 Clostridium pasteuranum dhaB P45514.1 1169287Citrobacter freundii dhaC AAB48851.1 1229154 Citrobacter freundii dhaEAAB48852.1 1229155 Citrobacter freundii

If a B 12-dependent diol dehydratase is utilized, heterologousexpression of the corresponding reactivating factor is recommended.B12-dependent diol dehydratases are subject to mechanism-based suicideactivation by substrates and some downstream products. Inactivation,caused by a tight association with inactive cobalamin, can be partiallyovercome by diol dehydratase reactivating factors in an ATP-dependentprocess. Regeneration of the B12 cofactor requires an additional ATP.Diol dehydratase regenerating factors are two-subunit proteins.Exemplary candidates are found in Klebsiella oxytoca (Mori et al., J.Biol. Chem. 272:32034-32041 (1997)), Salmonella typhimurium (Bobik etal., J. Bacteriol. 179:6633-6639 (1997); Chen et al., J. Bacteriol.176:5474-5482 (1994)), Lactobacillus collinoides (Sauvageot et al., FEMSMicrobiol. Lett. 209:69-74 (2002)), and Klebsiella pneumonia (WO2008/137403).

GenBank Gene Accession No. GI No. Organism ddrA AAC15871.1 3115376Klebsiella oxytoca ddrB AAC15872.1 3115377 Klebsiella oxytoca pduGAAL20947.1 16420573 Salmonella typhimurium pduH AAL20948.1 16420574Salmonella typhimurium pduG YP_002236779 206579698 Klebsiella pneumoniapduH YP_002236778 206579863 Klebsiella pneumonia pduG CAD01092 29335724Lactobacillus collinoides pduH CAD01093 29335725 Lactobacilluscollinoides

B 12-independent diol dehydratase enzymes utilize S-adenosylmethionine(SAM) as a cofactor, function under strictly anaerobic conditions, andrequire activation by a specific activating enzyme (Frey et al., Chem.Rev. 103:2129-2148 (2003)). The glycerol dehydrogenase and correspondingactivating factor of Clostridium butyricum, encoded by dhaB1 and dhaB2,have been well-characterized (O'Brien et al., Biochemistry 43:4635-4645(2004); Raynaud et al., Proc. Natl. Acad. Sci USA 100:5010-5015 (2003)).This enzyme was recently employed in a 1,3-propanediol overproducingstrain of E. coli and was able to achieve very high titers of product(Tang et al., Appl. Environ. Microbiol. 75:1628-1634(2009)(doi:AEM.02376-08, pii;10.1128/AEM.02376-08, doi). An additional B12-independent diol dehydratase enzyme and activating factor fromRoseburia inulinivorans was shown to catalyze the conversion of2,3-butanediol to 2-butanone (US publication 2009/09155870).

GenBank Gene Accession No. GI No. Organism dhaB1 AAM54728.1 27461255Clostridium butyricum dhaB2 AAM54729.1 27461256 Clostridium butyricumrdhtA ABC25539.1 83596382 Roseburia inulinivorans rdhtB ABC25540.183596383 Roseburia inulinivorans

Dihydroxy-acid dehydratase (DHAD, EC 4.2.1.9) is a B12-independentenzyme participating in branched-chain amino acid biosynthesis. In itsnative role, it converts 2,3-dihydroxy-3-methylvalerate to2-keto-3-methyl-valerate, a precursor of isoleucine. In valinebiosynthesis, the enzyme catalyzes the dehydration of2,3-dihydroxy-isovalerate to 2-oxoisovalerate. The DHAD from Sulfolobussolfataricus has a broad substrate range, and activity of a recombinantenzyme expressed in E. coli was demonstrated on a variety of aldonicacids (Kim and Lee, J. Biochem. 139:591-596 (2006)(doi:139/3/591,pii;10.1093/jb/mvj057, doi). The S. solfataricus enzyme is tolerant ofoxygen unlike many diol dehydratase enzymes. The E. coli enzyme, encodedby ilvD, is sensitive to oxygen, which inactivates its iron-sulfurcluster (Flint et al., J. Biol. Chem. 268:14732-14742 (1993)). Similarenzymes have been characterized in Neurospora crassa (Altmiller andWagner, Arch. Biochem. Biophys. 138:160-170 (1970)) and Salmonellatyphimurium (Armstrong et al., Biochim. Biophys. Acta 498:282-293(1977)).

GenBank Gene Accession No. GI No. Organism ilvD NP_344419.1 15899814Sulfolobus solfataricus ilvD AAT48208.1 48994964 Escherichia coli ilvDNP_462795.1 16767180 Salmonella typhimurium ilvD XP_958280.1 85090149Neurospora crassa

The diol dehydratase myo-inosose-2-dehydratase (EC 4.2.1.44) is anotherexemplary candidate. Myo-inosose is a six-membered ring containingadjacent alcohol groups. A purified enzyme encodingmyo-inosose-2-dehydratase functionality has been studied in Klebsiellaaerogenes in the context of myo-inositol degradation (Berman andMagasanik, J. Biol. Chem. 241:800-806 (1966)), but has not beenassociated with a gene to date. The myo-inosose-2-dehydratase ofSinorhizobium fredii was cloned and functionally expressed in E. coli(Yoshida et al., Biosci. Biotechnol. Biochem. 70:2957-2964(2006)(doi:JST.JSTAGE/bbb/60362, pii). A similar enzyme from B.subtilis, encoded by iolE, has also been studied (Yoshida et al.,Microbiology 150:571-580 (2004)).

GenBank Gene Accession No. GI No. Organism ME P42416.1 1176989 Bacillussubtilis ME AAX24114.1 60549621 Sinorhizobium fredii

EXAMPLE II Exemplary Pathway for Synthesis of p-Toluate from(2-Hydroxy-3-methyl-4-oxobutoxy)phosphonate by Shikimate Pathway Enzymes

This example describes exemplary pathways for synthesis of p-toluateusing shikimate pathway enzymes.

The chemical structure of p-toluate closely resembles p-hydroxybenzoate,a precursor of the electron carrier ubiquinone. 4-Hydroxybenzoate issynthesized from central metabolic precursors by enzymes in theshikimate pathway, found in bacteria, plants and fungi. The shikimatepathway is comprised of seven enzymatic steps that transformD-erythrose-4-phosphate (E4P) and phosphoenolpyruvate (PEP) tochorismate. Pathway enzymes include 2-dehydro-3-deoxyphosphoheptonate(DAHP) synthase, dehydroquinate (DHQ) synthase, DHQ dehydratase,shikimate dehydrogenase, shikimate kinase,5-enolpyruvylshikimate-3-phosphate (EPSP) synthase and chorismatesynthase. In the first step of the pathway, D-erythrose-4-phosphate andphosphoenolpyruvate are joined by DAHP synthase to form3-deoxy-D-arabino-heptulosonate-7-phosphate. This compound is thendephosphorylated, dehydrated and reduced to form shikimate. Shikimate isconverted to chorismate by the actions of three enzymes: shikimatekinase, 3-phosphoshikimate-2-carboxyvinyltransferase and chorismatesynthase. Subsequent conversion of chorismate to 4-hydroxybenzoate iscatalyzed by chorismate lyase.

The synthesis of p-toluate proceeds in an analogous manner as shown inFIG. 2. The pathway originates with PEP and 2H3M4OP, a compoundanalogous to E4P with a methyl group in place of the 3-hydroxyl group ofE4P. The hydroxyl group of E4P does not directly participate in thechemistry of the shikimate pathway reactions, so the methyl-substituted2H3M4OP precursor is expected to react as an alternate substrate.Directed or adaptive evolution can be used to improve preference for2H3M4OP and downstream derivatives as substrates. Such methods arewell-known in the art.

Strain engineering strategies for improving the efficiency of fluxthrough shikimate pathway enzymes are also applicable here. Theavailability of the pathway precursor PEP can be increased by alteringglucose transport systems (Yi et al., Biotechnol. Prog. 19:1450-1459(2003)(doi:10.1021/bp0340584, doi). 4-Hydroxybenzoate-overproducingstrains were engineered to improve flux through the shikimate pathway bymeans of overexpression of a feedback-insensitive isozyme of3-deoxy-D-arabinoheptulosonic acid-7-phosphate synthase (Barker andFrost, Biotechnol. Bioeng. 76:376-390 (2001)(doi:10.1002/bit.10160,pii). Additionally, expression levels of shikimate pathway enzymes andchorismate lyase were enhanced. Similar strategies can be employed in astrain for overproducing p-toluate.

A. 2-Dehydro-3-deoxyphosphoheptonate synthase (EC 2.5.1.54). Thecondensation of D-erythrose-4-phosphate and phosphoenolpyruvate iscatalyzed by 2-dehydro-3-deoxyphosphoheptonate (DAHP) synthase (EC2.5.1.54). Three isozymes of this enzyme are encoded in the E. coligenome by aroG, aroF and aroH and are subject to feedback inhibition byphenylalanine, tyrosine and tryptophan, respectively. In wild-type cellsgrown on minimal medium, the aroG, aroF and aroH gene productscontributed 80%, 20% and 1% of DAHP synthase activity, respectively(Hudson and Davidson, J. Mol. Biol. 180:1023-1051(1984)(doi:0022-2836(84)90269-9, pii). Two residues of AroG were foundto relieve inhibition by phenylalanine (Kikuchi et al., Appl. Environ.Microbiol. 63:761-762 (1997)). The feedback inhibition of AroF bytyrosine was removed by a single base-pair change (Weaver and Herrmann,J. Bacteriol. 172:6581-6584 (1990)). The tyrosine-insensitive DAHPsynthase was overexpressed in a 4-hydroxybenzoate-overproducing strainof E. coli (Barker and Frost, Biotechnol. Bioeng. 76:376-390(2001)(doi:10.1002/bit.10160, pii). The aroG gene product was shown toaccept a variety of alternate 4- and 5-carbon length substrates(Sheflyan et al., J. Am. Chem. Soc. 120(43):11027-11032 (1998);Williamson et al., Bioorg. Med. Chem. Lett. 15:2339-2342(2005)(doi:S0960-894X(05)00273-8, pii;10.1016/j.bmc1.2005.02.080, doi).The enzyme reacts efficiently with (3S)-2-deoxyerythrose-4-phosphate, asubstrate analogous to D-erythrose-4-phosphate but lacking the alcoholat the 2-position (Williamson et al., supra 2005). Enzymes fromHelicobacter pylori and Pyrococcus furiosus also accept this alternatesubstrate (Schofield et al., Biochemistry 44:11950-11962(2005)(doi:10.1021/bi050577z, doi; Webby et al., Biochem. J. 390:223-2302005)(doi:BJ20050259, pii;10.1042/BJ20050259, doi) and have beenexpressed in E. coli. An evolved variant of DAHP synthase, differingfrom the wild type E. coli AroG enzyme by 7 amino acids, was shown toexhibit a 60-fold improvement in Kcat/K_(M) (Ran and Frost, J. Am. Chem.Soc. 129:6130-6139 (2007)(doi:10.1021/ja067330p, doi).

GenBank Gene Accession No. GI No. Organism aroG AAC73841.1 1786969Escherichia coli aroF AAC75650.1 1788953 Escherichia coli aroHAAC74774.1 1787996 Escherichia coli aroF Q9ZMU5 81555637 Helicobacterpylori PF1690 NP_579419.1 18978062 Pyrococcus furiosus

B. 3-Dehydroquinate synthase (EC 4.2.3.4). The dephosphorylation ofsubstrate(2)(2,4-dihydroxy-5-methyl-6-[(phosphonooxy)methyl]oxane-2-carboxylate)to substrate (3)(1,3-dihydroxy-4-methylcylohex-1-ene-1-carboxylate) asshown in FIG. 2 is analogous to the dephosphorylation of3-deoxy-arabino-heptulonate-7-phosphate by 3-dehydroquinate synthase.The enzyme has been characterized in E. coli (Mehdi et al., MethodsEnzymol. 142:306-314 (1987), B. subtilis (Hasan and Nester, J. Biol.Chem. 253:4999-5004 (1978)) and Mycobacterium tuberculosis H3 7Rv (deMendonca et al., J. Bacteriol. 189:6246-6252 (2007)(doi:JB.00425-07,pii;10.1128/JB.00425-07, doi). The E. coli enzyme is subject toinhibition by L-tyrosine (Barker and Frost, Biotechnol. Bioeng.76:376-390 2001)(doi:10.1002/bit.10160, pii).

GenBank Gene Accession No. GI No. Organism aroB AAC76414.1 1789791Escherichia coli aroB NP_390151.1 16079327 Bacillus subtilis aroBCAB06200.1 1781064 Mycobacterium tuberculosis

C. 3-Dehydroquinate dehydratase (EC 4.2.1.10). 3-Dehydroquinatedehydratase, also termed 3-dehydroquinase (DHQase), naturally catalyzesthe dehydration of 3-dehydroquinate to 3-dehydroshikimate, analogous tostep C in the p-toluate pathway of FIG. 2. DHQase enzymes can be dividedinto two classes based on mechanism, stereochemistry and sequencehomology (Gourley et al., Nat. Struct. Biol. 6:521-525.(1999)(doi:10.1038/9287, doi). Generally the type 1 enzymes are involvedin biosynthesis, while the type 2 enzymes operate in the reverse(degradative) direction. Type 1 enzymes from E. coli (Kinghorn et al.,Gene 14:73-80. 1981)(doi:0378-1119(81)90149-9, pii), Salmonella typhi(Kinghorn et al., supra 1981; Servos et al., J. Gen. Microbiol.137:147-152 (1991)) and B. subtilis (Warburg et al., Gene 32:57-661984)(doi:0378-1119(84)90032-5, pii) have been cloned and characterized.Exemplary type II 3-dehydroquinate dehydratase enzymes are found inMycobacterium tuberculosis, Streptomyces coelicolor (Evans et al., FEBSLett. 530:24-30 (2002)) and Helicobacter pylori (Lee et al., Proteins51:616-7 (2003)).

GenBank Gene Accession No. GI No. Organism aroD AAC74763.1 1787984Escherichia coli aroD P24670.2 17433709 Salmonella typhi aroCNP_390189.1 16079365 Bacillus subtilis aroD P0A4Z6.2 61219243Mycobacterium tuberculosis aroQ P15474.3 8039781 Streptomyces coelicoloraroQ Q48255.2 2492957 Helicobacter pylori

D. Shikimate dehydrogenase (EC 1.1.1.25). Shikimate dehydrogenasecatalyzes the NAD(P)H dependent reduction of 3-dehydroshikimate toshikimate, analogous to Step D of FIG. 2. The E. coli genome encodes twoshikimate dehydrogenase paralogs with different cofactor specificities.The enzyme encoded by aroE is NADPH specific, whereas the ydiB geneproduct is a quinate/shikimate dehydrogenase which can utilize NADH(preferred) or NADPH as a cofactor (Michel et al., J. Biol. Chem.278:19463-19472 (2003)(doi:10.1074/jbc.M300794200, doi;M300794200, pii).NADPH-dependent enzymes from Mycobacterium tuberculosis (Zhang et al.,J. Biochem. Mol. Biol. 38:624-631 (2005)), Haemophilus influenzae (Ye etal., J. Bacteriol. 185:4144-4151 (2003)) and Helicobacter pylori (Han etal., FEBS J. 273:4682-4692 (2006)(doi:EJB5469,pii;10.1111/j.1742-4658.2006.05469.x, doi) have been functionallyexpressed in E. coli.

GenBank Gene Accession No. GI No. Organism aroE AAC76306.1 1789675Escherichia coli ydiB AAC74762.1 1787983 Escherichia coli aroENP_217068.1 15609689 Mycobacterium tuberculosis aroE P43876.1 1168510Haemophilus influenzae aroE AAW22052.1 56684731 Helicobacter pylori

E. Shikimate kinase (EC 2.7.1.71). Shikimate kinase catalyzes theATP-dependent phosphorylation of the 3-hydroxyl group of shikimateanalogous to Step E of FIG. 2. Two shikimate kinase enzymes are encodedby aroK (SKI) and aroL (SK2) in E. coli (DeFeyter and Pittard, J.Bacteriol. 165:331-333 (1986); Lobner-Olesen and Marinus, J. Bacteriol.174:525-529 (1992)). The Km of SK2, encoded by aroL, is 100-fold lowerthan that of SK1, indicating that this enzyme is responsible foraromatic biosynthesis (DeFeyter et al., supra 1986). Additionalshikimate kinase enzymes from Mycobacterium tuberculosis (Gu et al., J.Mol. Biol. 319:779-789 (2002)(doi:10.1016/S0022-2836(02)00339-X,doi;S0022-2836(02)00339-X, pii) Oliveira et al., Protein Expr. Purif.22:430-435 (2001)(doi:10.1006/prep.2001.1457, doi;S1046-5928(01)91457-3,pii), Helicobacter pylori (Cheng et al., J. Bacterial. 187:8156-8163(2005)(doi:187/23/8156, pii;10.1128/JB.187.23.8156-8163.2005, doi) andErwinia chrysanthemi (Krell et al., Protein Sci. 10:1137-1149(2001)(doi:10.1110/ps.52501, doi) have been cloned in E. coli.

GenBank Gene Accession No. GI No. Organism aroK YP_026215.2 90111581Escherichia coli aroL NP_414922.1 16128373 Escherichia coli aroKCAB06199.1 1781063 Mycobacterium tuberculosis aroK NP_206956.1 15644786Helicobacter pylori SK CAA32883.1 42966 Erwinia chrysanthemi

F. 3-Phosphoshikimate-2-carboxyvinyltransferase (EC 2.5.1.19).3-Phosphoshikimate-2-carboxyvinyltransferase, also known as5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), catalyzes thetransfer of the enolpyruvyl moiety of phosphoenolpyruvate to the5-hydroxyl of shikimate-3-phosphate. The enzyme is encoded by aroA in E.coli (Anderson et al., Biochemistry 27:1604-1610 (1988)). EPSPS enzymesfrom Mycobacterium tuberculosis (Oliveira et al., Protein Expr. Purif.22:430-435 (2001)(doi:10.1006/prep.2001.1457, doi;S1046-5928(01)91457-3,pii), Dunaliella salina (Yi et al., J. Microbiol. 45:153-157(2007)(doi:2519, pii) and Staphylococcus aureus (Priestman et al., FEBSLett. 579:728-732 (2005)(doi:S0014-5793(05)00012-8,pii;10.1016/j.febslet.2004.12.057, doi) have been cloned andfunctionally expressed in E. coli.

GenBank Gene Accession No. GI No. Organism aroA AAC73994.1 1787137Escherichia coli aroA AAA25356.1 149928 Mycobacterium tuberculosis aroAAAA71897.1 152956 Staphylococcus aureus aroA ABM68632.1 122937807Dunaliella salina

G. Chorismate synthase (EC 4.2.3.5). Chorismate synthase is the seventhenzyme in the shikimate pathway, catalyzing the transformation of5-enolpyruvylshikimate-3-phosphate to chorismate. The enzyme requiresreduced flavin mononucleotide (FMN) as a cofactor, although the netreaction of the enzyme does not involve a redox change. In contrast tothe enzyme found in plants and bacteria, the chorismate synthase infungi is also able to reduce FMN at the expense of NADPH (Macheroux etal., Planta 207:325-334 (1999)). Representative monofunctional enzymesare encoded by aroC of E. coli (White et al., Biochem. J. 251:313-322(1988)) and Streptococcus pneumoniae (Maclean and Ali, Structure11:1499-1511 (2003)(doi:S0969212603002648, pii). Bifunctional fungalenzymes are found in Neurospora crassa (Kitzing et al., J. Biol. Chem.276:42658-42666 (2001)(doi:10.1074/jbc.M107249200, doi;M107249200, pii)and Saccharomyces cerevisiae (Jones et al., Mol. Microbiol. 5:2143-2152(1991)).

GenBank Gene Accession No. GI No. Organism aroC NP_416832.1 16130264Escherichia coli aroC ACH47980.1 197205483 Streptococcus pneumoniaeU25818.1: AAC49056.1 976375 Neurospora crassa 19 . . . 1317 ARO2CAA42745.1 3387 Saccharomyces cerevisiae

H. Chorismate lyase (EC 4.1.3.40). Chorismate lyase catalyzes the firstcommitted step in ubiquinone biosynthesis: the removal of pyruvate fromchorismate to form 4-hydroxybenzoate. The enzymatic reaction israte-limited by the slow release of the 4-hydroxybenzoate product(Gallagher et al., Proteins 44:304-311 (2001)(doi:10.1002/prot.1095,pii), which is thought to play a role in delivery of 4-hydroxybenzoateto downstream membrane-bound enzymes. The chorismate lyase of E. coliwas cloned and characterized and the enzyme has been crystallized(Gallagher et al., supra 2001; Siebert et al., FEBS Lett. 307:347-350(1992)(doi:0014-5793(92)80710-X, pii). Structural studies implicate theG90 residue as contributing to product inhibition (Smith et al., Arch.Biochem. Biophys. 445:72-80 (2006)(doi:S0003-9861(05)00446-7,pii;10.1016/j.abb.2005.10.026, doi). Modification of two surface-activecysteine residues reduced protein aggregation (Holden et al., Biochim.Biophys. Acta 1594:160-167 (2002)(doi:S0167483801003028, pii). Arecombinant form of the Mycobacterium tuberculosis chorismate lyase wascloned and characterized in E. coli (Stadthagen et al., J. Biol. Chem.280:40699-40706 2005)(doi:M508332200, pii;10.1074/jbc.M508332200, doi).

GenBank Gene Accession No. GI No. Organism ubiC AAC77009.2 87082361Escherichia coli Rv2949c NP_217465.1 15610086 Mycobacterium tuberculosis

B-F. Multifunctional AROM protein. In most bacteria, the enzymes of theshikimate pathway are encoded by separate polypeptides. In microbialeukaryotes, five enzymatic functions are catalyzed by a polyfunctionalprotein encoded by a pentafunctional supergene (Campbell et al., Int. J.Parasitol. 34:5-13 (2004)(doi:S0020751903003102, pii). Themultifunctional AROM protein complex catalyzes reactions analogous toreactions B-F of FIG. 2. The AROM protein complex has been characterizedin fungi including Aspergillus nidulans, Neurospora crassa,Saccharomyces cerevisiae and Pneumocystis carinii (Banerji et al., J.Gen. Microbiol. 139:2901-2914 (1993); Charles et al., Nucleic Acids Res.14:2201-2213 (1986); Coggins et al., Methods Enzymol. 142:325-341(1987); Duncan, K., Biochem. J. 246:375-386 (1987)). Several componentsof AROM have been shown to function independently as individualpolypeptides. For example, dehydroquinate synthase (DHQS) forms theamino-terminal domain of AROM, and can function independently whencloned into E. coli (Moore et al., Biochem. J. 301 (Pt 1):297-304(1994)). Several crystal structures of AROM components from Aspergillusnidulans provide insight into the catalytic mechanism (Carpenter et al.,Nature 394:299-302 (1998)(doi:10.1038/28431, doi).

GenBank Gene Accession No. GI No. Organism AROM P07547.3 238054389Aspergillus nidulans AROM P08566.1 114166 Saccharomyces cerevisiae AROMP07547.3 238054389 Aspergillus nidulans AROM Q12659.1 2492977Pneumocystis carinii

EXAMPLE III Exemplary Pathway for Enzymatic Transformation of p-Toluateto Terephthalic Acid

This example describes exemplary pathways for conversion of p-toluate toterephthalic acid (PTA).

P-toluate can be further transformed to PTA by oxidation of the methylgroup to an acid in three enzymatic steps as shown in FIG. 3. Thepathway is comprised of a p-toluate methyl-monooxygenase reductase, a4-carboxybenzyl alcohol dehydrogenase and a 4-carboxybenzyl aldehydedehydrogenase. In the first step, p-toluate methyl-monooxyngenaseoxidizes p-toluate to 4-carboxybenzyl alcohol in the presence of O₂. TheComamonas testosteroni enzyme (tsaBM), which also reacts with 4-toluenesulfonate as a substrate, has been purified and characterized (Locher etal., J. Bacteriol. 173:3741-3748 (1991)). 4-Carboxybenzyl alcohol issubsequently converted to an aldehyde by 4-carboxybenzyl alcoholdehydrogenase (tsaC). The aldehyde to acid transformation is catalyzedby 4-carboxybenzaldehyde dehydrogenase (tsaD). Enzymes catalyzing thesereactions are found in Comamonas testosteroni T-2, an organism capableof utilizing p-toluate as the sole source of carbon and energy (Junkeret al., J. Bacteriol. 179:919-927 (1997)). Additional genes to transformp-toluate to PTA can be found by sequence homology, in particular toproteobacteria in the genera Burkholderia, Alcaligenes, Pseudomonas,Shingomonas and Comamonas (U.S. Pat. No. 6,187,569 and US publication2003/0170836). Genbank identifiers associated with the Comamonastestosteroni enzymes are listed below.

GenBank Gene Accession No. GI No. Organism tsaB AAC44805.1 1790868Comamonas testosteroni tsaM AAC44804.1 1790867 Comamonas testosteronitsaC AAC44807.1 1790870 Comamonas testosteroni tsaD AAC44808.1 1790871Comamonas testosteroni

Throughout this application various publications have been referenced.The disclosures of these publications in their entireties, includingGenBank and GI number publications, are hereby incorporated by referencein this application in order to more fully describe the state of the artto which this invention pertains. Although the invention has beendescribed with reference to the examples provided above, it should beunderstood that various modifications can be made without departing fromthe spirit of the invention.

1. A non-naturally occurring microbial organism, comprising a microbialorganism having a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathwaycomprising at least one exogenous nucleic acid encoding a(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway enzyme expressed ina sufficient amount to produce(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, said(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway comprising2-C-methyl-D-erythritol-4-phosphate dehydratase.
 2. The non-naturallyoccurring microbial organism of claim 1, wherein said(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway further comprises1-deoxyxylulose-5-phosphate synthase or 1-deoxy-D-xylulose-5-phosphatereductoisomerase.
 3. The non-naturally occurring microbial organism ofclaim 1, wherein said (2-hydroxy-3-methyl-4-oxobutoxy)phosphonatepathway further comprises 1-deoxyxylulose-5-phosphate synthase and1-deoxy-D-xylulose-5-phosphate reductoisomerase.
 4. The non-naturallyoccurring microbial organism of claim 1, wherein said microbial organismcomprises three exogenous nucleic acids each encoding a(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway enzyme.
 5. Thenon-naturally occurring microbial organism of claim 4, wherein saidthree exogenous nucleic acids encode 2-C-methyl-D-erythritol-4-phosphatedehydratase, 1-deoxyxylulose-5-phosphate synthase and1-deoxy-D-xylulose-5-phosphate reductoisomerase.
 6. The non-naturallyoccurring microbial organism of claim 1, wherein said at least oneexogenous nucleic acid is a heterologous nucleic acid.
 7. Thenon-naturally occurring microbial organism of claim 1, wherein saidnon-naturally occurring microbial organism is in a substantiallyanaerobic culture medium.
 8. A method for producing(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, comprising culturing thenon-naturally occurring microbial organism of claim 1 under conditionsand for a sufficient period of time to produce(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate.
 9. The method of claim 8,wherein said non-naturally occurring microbial organism is in asubstantially anaerobic culture medium.
 10. The method of claim 8,wherein said microbial organism comprises three exogenous nucleic acidseach encoding a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathwayenzyme.
 11. The method of claim 10, wherein said three exogenous nucleicacids encode 2-C-methyl-D-erythritol-4-phosphate dehydratase,1-deoxyxylulose-5-phosphate synthase and 1-deoxy-D-xylulose-5-phosphatereductoisomerase.
 12. The method of claim 8, wherein said at least oneexogenous nucleic acid is a heterologous nucleic acid.
 13. Anon-naturally occurring microbial organism, comprising a microbialorganism having a p-toluate pathway comprising at least one exogenousnucleic acid encoding a p-toluate pathway enzyme expressed in asufficient amount to produce p-toluate, said p-toluate pathwaycomprising 2-dehydro-3-deoxyphosphoheptonate synthase; 3-dehydroquinatesynthase; 3-dehydroquinate dehydratase; shikimate dehydrogenase;shikimate kinase; 3-phosphoshikimate-2-carboxyvinyltransferase;chorismate synthase; or chorismate lyase.
 14. The non-naturallyoccurring microbial organism of claim 13, wherein said microbialorganism comprises two exogenous nucleic acids each encoding a p-toluatepathway enzyme.
 15. The non-naturally occurring microbial organism ofclaim 13, wherein said microbial organism comprises three exogenousnucleic acids each encoding a p-toluate pathway enzyme.
 16. Thenon-naturally occurring microbial organism of claim 13, wherein saidmicrobial organism comprises four exogenous nucleic acids each encodinga p-toluate pathway enzyme.
 17. The non-naturally occurring microbialorganism of claim 13, wherein said microbial organism comprises fiveexogenous nucleic acids each encoding a p-toluate pathway enzyme. 18.The non-naturally occurring microbial organism of claim 13, wherein saidmicrobial organism comprises six exogenous nucleic acids each encoding ap-toluate pathway enzyme.
 19. The non-naturally occurring microbialorganism of claim 13, wherein said microbial organism comprises sevenexogenous nucleic acids each encoding a p-toluate pathway enzyme. 20.The non-naturally occurring microbial organism of claim 13, wherein saidmicrobial organism comprises seven exogenous nucleic acids each encodinga p-toluate pathway enzyme.
 21. The non-naturally occurring microbialorganism of claim 19, wherein said eight exogenous nucleic acids encode2-dehydro-3-deoxyphosphoheptonate synthase; 3-dehydroquinate synthase;3-dehydroquinate dehydratase; shikimate dehydrogenase; shikimate kinase;3-phosphoshikimate-2-carboxyvinyltransferase; chorismate synthase; andchorismate lyase.
 22. The non-naturally occurring microbial organism ofclaim 13, wherein said microbial organism further comprises a(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway.
 23. Thenon-naturally occurring microbial organism of claim 22, wherein the(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway comprises2-C-methyl-D-erythritol-4-phosphate dehydratase,1-deoxyxylulose-5-phosphate synthase or 1-deoxy-D-xylulose-5-phosphatereductoisomerase.
 24. The non-naturally occurring microbial organism ofclaim 23, wherein the (2-hydroxy-3-methyl-4-oxobutoxy)phosphonatepathway comprises 2-C-methyl-D-erythritol-4-phosphate dehydratase,1-deoxyxylulose-5-phosphate synthase and 1-deoxy-D-xylulose-5-phosphatereductoisomerase.
 25. The non-naturally occurring microbial organism ofclaim 13, wherein at least one exogenous nucleic acid is a heterologousnucleic acid.
 26. The non-naturally occurring microbial organism ofclaim 13, wherein said non-naturally occurring microbial organism is ina substantially anaerobic culture medium.
 27. A method for producingp-toluate, comprising culturing the non-naturally occurring microbialorganism of claim 13 under conditions and for a sufficient period oftime to produce p-toluate.
 28. The method of claim 27, wherein saidnon-naturally occurring microbial organism is in a substantiallyanaerobic culture medium.
 29. The method of claim 27, wherein saidmicrobial organism comprises seven exogenous nucleic acids each encodinga p-toluate pathway enzyme.
 30. The method of claim 29, wherein saidseven exogenous nucleic acids encode 2-dehydro-3-deoxyphosphoheptonatesynthase; 3-dehydroquinate synthase; 3-dehydroquinate dehydratase;shikimate dehydrogenase; shikimate kinase;3-phosphoshikimate-2-carboxyvinyltransferase; chorismate synthase; andchorismate lyase.
 31. The method of claim 27, wherein said microbialorganism further comprises a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonatepathway.
 32. The method of claim 31, wherein the(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway comprises2-C-methyl-D-erythritol-4-phosphate dehydratase,1-deoxyxylulose-5-phosphate synthase or 1-deoxy-D-xylulose-5-phosphatereductoisomerase.
 33. The method of claim 32, wherein the(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway comprises2-C-methyl-D-erythritol-4-phosphate dehydratase,1-deoxyxylulose-5-phosphate synthase and 1-deoxy-D-xylulose-5-phosphatereductoisomerase.
 34. The method of claim 27, wherein said at least oneexogenous nucleic acid is a heterologous nucleic acid.
 35. Anon-naturally occurring microbial organism, comprising a microbialorganism having a terephthalate pathway comprising at least oneexogenous nucleic acid encoding a terephthalate pathway enzyme expressedin a sufficient amount to produce terephthalate, said terephthalatepathway comprising p-toluate methyl-monooxygenase reductase;4-carboxybenzyl alcohol dehydrogenase; or 4-carboxybenzyl aldehydedehydrogenase; and wherein said microbial organism further comprises ap-toluate pathway, wherein said p-toluate pathway comprises2-dehydro-3-deoxyphosphoheptonate synthase; 3-dehydroquinate synthase;3-dehydroquinate dehydratase; shikimate dehydrogenase; shikimate kinase;3-phosphoshikimate-2-carboxyvinyltransferase; chorismate synthase; orchorismate lyase.
 36. The non-naturally occurring microbial organism ofclaim 35, wherein said microbial organism comprises two exogenousnucleic acids each encoding a terephthalate pathway enzyme.
 37. Thenon-naturally occurring microbial organism of claim 35, wherein saidmicrobial organism comprises three exogenous nucleic acids each encodinga terephthalate pathway enzyme.
 38. The non-naturally occurringmicrobial organism of claim 37, wherein said three exogenous nucleicacids encode p-toluate methyl-monooxygenase reductase; 4-carboxybenzylalcohol dehydrogenase; and 4-carboxybenzyl aldehyde dehydrogenase. 39.The non-naturally occurring microbial organism of claim 35, wherein saidp-toluate pathway comprises 2-dehydro-3-deoxyphosphoheptonate synthase;3-dehydroquinate synthase; 3-dehydroquinate dehydratase; shikimatedehydrogenase; shikimate kinase;3-phosphoshikimate-2-carboxyvinyltransferase; chorismate synthase; andchorismate lyase.
 40. The non-naturally occurring microbial organism ofclaim 35, wherein said microbial organism further comprises a(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway.
 41. Thenon-naturally occurring microbial organism of claim 40, wherein the(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway comprises2-C-methyl-D-erythritol-4-phosphate dehydratase,1-deoxyxylulose-5-phosphate synthase or 1-deoxy-D-xylulose-5-phosphatereductoisomerase.
 42. The non-naturally occurring microbial organism ofclaim 41, wherein the (2-hydroxy-3-methyl-4-oxobutoxy)phosphonatepathway comprises 2-C-methyl-D-erythritol-4-phosphate dehydratase,1-deoxyxylulose-5-phosphate synthase and 1-deoxy-D-xylulose-5-phosphatereductoisomerase.
 43. The non-naturally occurring microbial organism ofclaim 35, wherein said at least one exogenous nucleic acid is aheterologous nucleic acid.
 44. The non-naturally occurring microbialorganism of claim 35, wherein said non-naturally occurring microbialorganism is in a substantially anaerobic culture medium.
 45. A methodfor producing terephthalate, comprising culturing the non-naturallyoccurring microbial organism of claim 35 under conditions and for asufficient period of time to produce terephthalate.
 46. The method ofclaim 45, wherein said non-naturally occurring microbial organism is ina substantially anaerobic culture medium.
 47. The method of claim 45,wherein said microbial organism comprises three exogenous nucleic acidseach encoding a terephthalate pathway enzyme.
 48. The method of claim47, wherein said three exogenous nucleic acids encode p-toluatemethyl-monooxygenase reductase; 4-carboxybenzyl alcohol dehydrogenase;or 4-carboxybenzyl aldehyde dehydrogenase.
 49. The method of claim 45,wherein said p-toluate pathway comprises2-dehydro-3-deoxyphosphoheptonate synthase; 3-dehydroquinate synthase;3-dehydroquinate dehydratase; shikimate dehydrogenase; shikimate kinase;3-phosphoshikimate-2-carboxyvinyltransferase; chorismate synthase; andchorismate lyase.
 50. The method of claim 45, wherein said microbialorganism further comprises a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonatepathway.
 51. The method of claim 50, wherein the(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway comprises2-C-methyl-D-erythritol-4-phosphate dehydratase,1-deoxyxylulose-5-phosphate synthase or 1-deoxy-D-xylulose-5-phosphatereductoisomerase.
 52. The method of claim 51, wherein the(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway comprises2-C-methyl-D-erythritol-4-phosphate dehydratase,1-deoxyxylulose-5-phosphate synthase and 1-deoxy-D-xylulose-5-phosphatereductoisomerase.
 53. The method of claim 45, wherein said at least oneexogenous nucleic acid is a heterologous nucleic acid.